Chronic wasting disease prions are not
transmissible to transgenic mice overexpressing
human prion protein
Malin K. Sandberg,1Huda Al-Doujaily,1Christina J. Sigurdson,2
Markus Glatzel,3Catherine O’Malley,1Caroline Powell,1
Emmanuel A. Asante,1Jacqueline M. Linehan,1Sebastian Brandner,1
Jonathan D. F. Wadsworth1and John Collinge1
Received 9 June 2010
Accepted 6 July 2010
1MRC Prion Unit and Department of Neurodegenerative Disease, UCL Institute of Neurology,
National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK
2Department of Pathology, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA
3Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52,
D-20246 Hamburg, Germany
Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids,
including mule deer, white-tailed deer, Rocky Mountain elk and moose. CWD-infected cervids
have been reported in 14 USA states, two Canadian provinces and in South Korea. The possibility
of a zoonotic transmission of CWD prions via diet is of particular concern in North America where
hunting of cervids is a popular sport. To investigate the potential public health risks posed by
CWD prions, we have investigated whether intracerebral inoculation of brain and spinal cord from
CWD-infected mule deer transmits prion infection to transgenic mice overexpressing human prion
protein with methionine or valine at polymorphic residue 129. These transgenic mice have been
utilized in extensive transmission studies of human and animal prion disease and are susceptible
to BSE and vCJD prions, allowing comparison with CWD. Here, we show that these mice proved
entirely resistant to infection with mule deer CWD prions arguing that the transmission barrier
associated with this prion strain/host combination is greater than that observed with classical BSE
prions. However, it is possible that CWD may be caused by multiple prion strains. Further studies
will be required to evaluate the transmission properties of distinct cervid prion strains as they are
Chronic wasting disease (CWD) is a prion disease affecting
free-ranging and captive cervids, including mule deer,
white-tailed deer, RockyMountain
(Williams & Young, 1980, 1982; Williams, 2005; Baeten
et al., 2007). Like all mammalian prion diseases, which
include Creutzfeldt–Jakob disease (CJD), kuru and variant
CJD (vCJD) in humans and bovine spongiform encephalo-
pathy (BSE) in cattle, the central event in CWD infection is
the post-translational conversion of the host-encoded,
cellular prion protein (PrPC), to an abnormal isoform,
designated PrPSc(Prusiner, 1998; Collinge & Clarke, 2007).
Progressive accumulation of PrPScin the central nervous
system (Guiroy et al., 1993) is associated with clinical signs
of CWD which include weight loss, behavioural changes,
excessive salivation, difficulty swallowing, polydipsia, poly-
uria, and ataxia prior to death (Williams & Young, 1980,
1982; Williams, 2005). International concern over CWD is
growing as infected cervids have now been reported in 14
states in North America, two Canadian provinces and in
South Korea (Kim et al., 2005; Williams, 2005; Sigurdson &
Aguzzi, 2007; Sigurdson, 2008). To date, CWD has not
been reported in Europe, although surveillance has been
The prevalence of CWD infection can reach levels of up to
30% in free-ranging herds in North America and up to 90%
in animals housed in CWD research facilities (Williams,
2005). Infectious prions in the saliva (Mathiason et al., 2006;
Haley et al., 2009; Mathiason et al., 2009), urine (Haley et al.,
2009) and faeces of CWD-infected animals (Tamguney et al.,
2009) may underlie the highly efficient natural transmission
of CWD among cervids through environmental contamina-
tion (Mathiason et al., 2009). Protease-resistant cervid prion
protein has recently been demonstrated in an environmental
water sample from a CWD endemic area (Nichols et al.,
Journal of General Virology (2010), 91, 2651–2657
024380G2010 SGMPrinted in Great Britain 2651
Despite efficient horizontal transmission of CWD prions
among cervids, to date there is no clear evidence for natural
disease transmission to other species. A recent survey for
transmissible spongiform encephalopathy in scavengers of
white-tailed deer carcasses in a CWD endemic area of
Wisconsin found no evidence for cross-species transmission
(Jennelle et al., 2009). Nevertheless, the zoonotic transmis-
sion of BSE prions (Collinge et al., 1996; Hill et al., 1997;
Bruce et al., 1997; Asante et al., 2002; Wadsworth &
Collinge, 2007) has dramatically highlighted the potential
risk posed to humans from dietary exposure to CWD prions
(Belay, 2004; Sigurdson, 2008). Infectious prions are present
in the blood (Mathiason et al., 2006), skeletal muscle
(Angers et al., 2006) and fat (Race et al., 2009a) of CWD-
infected deer and CWD prions have been shown to be
deer and cervid PrP expressing transgenic mice (Hamir
et al., 2006; Fox et al., 2006; Trifilo et al., 2007).
Consumption of hunted deer and elk is widely practised in
North America and a survey conducted by the American
Red Cross and other blood banking establishments has
reported that ~40% of USA blood donors have consumed
venison obtained from the wild (Belay et al., 2001). To date,
however, epidemiological surveillance has not indicated any
link between human disease and CWD exposure (Belay,
2004; Mawhinney et al., 2006; Anderson et al., 2007).
However, incubation periods in human prion disease even
in the absence of a transmission barrier can exceed 50 years
(Collinge et al., 2006, 2008). Accordingly, there has been
intense research interest in establishing the host range of
CWD prions through experimental transmissions to labor-
atory animals (Tamguney et al., 2006; Raymond et al., 2007;
Sigurdson et al., 2008; Heisey et al., 2010) and through the
use of in vitro prion amplification systems (Raymond et al.,
2000; Kurt et al., 2009).
Concern that CWD prions might be transmissible to
humans was heightened in 2005 by the finding that squirrel
monkeys can be infected by intracerebral inoculation with
CWD mule deer brain homogenate (Marsh et al., 2005).
However, a more recent study has shown that cynomolgus
macaques (that are evolutionarily closer to humans) differ
significantly from squirrel monkeys with respect to their
susceptibility to infection with CWD prions, with no
evidence for clinical disease in macaques at 70 months
post-inoculation (Race et al., 2009b). Crucially however,
because prion transmission barriers and prion strains are
intimately related by conformational selection (Collinge,
1999; Collinge & Clarke, 2007) the ability of CWD prions
to propagate in humans cannot be inferred by studying the
interaction of CWD prions with distinct (albeit highly
conserved) PrP sequences from other species. To date, two
studies have reported that transgenic mice expressing
human PrP with methionine at polymorphic residue 129
are resistant to intracerebral challenge with CWD prions.
The first of these studies used two lines of transgenic mice
expressing human PrP at either one or two times the
endogenous level of mouse brain. After inoculation with
CWD-infected elk brain homogenate, none of these
transgenic mice showed clinical signs of prion disease or
detectable accumulation of abnormal PrP by either
immunohistochemistry or immunoblotting (Kong et al.,
2005). Although these mice are susceptible to infection
with atypical BSE prions, their susceptibility to classic BSE
prions or vCJD prions has not been reported (Kong et al.,
2008). The second study used hemizygous transgenic mice
expressing human PrP at two times the endogenous level of
murine PrP expression in mouse brain. No evidence of
clinical prion disease was observed following intracerebral
challenge with CWD-infected elk, mule deer or white-
tailed deer brain homogenate; however, importantly
subclinical infection was not excluded (Tamguney et al.,
2006). Susceptibility of these mice to infection with BSE or
vCJD prions has not been reported. Here, to investigate
further the potential risks for transmission of cervid prions
to humans, we have transmitted mule deer CWD prions to
lines of transgenic mice overexpressing human PrP two- to
sixfold with either methionine or valine at polymorphic
residue 129 in which we have extensive experience of
transmission of a wide range of human acquired, sporadic
and inherited prion disease isolates, including kuru and
multiple vCJD cases (Collinge et al., 1995a, b, 1996; Hill
et al., 1997; Wadsworth et al., 2008a). Extensive compar-
ative data are available on transmission of multiple cattle
BSE isolates (Hill et al., 1997; Asante et al., 2002, 2006;
Wadsworth et al., 2004) as well as BSE experimentally
passaged or naturally transmitted to multiple mammalian
species and these mice are therefore suitable for compar-
ative assessment of the zoonotic potential of CWD prions.
Immunoblot analysis of CWD-infected brain and
CWD-infected mule deer brain (from animal D10) and
spinal cord (from animal D08) originated from captive
Research Centre, Colorado, USA. Homogenates (10% w/v)
of these tissues were prepared in PBS and examined for
proteinase K (PK)-resistant PrP by immunoblotting. Both
samples showed a high level of cervid PrPSc(Fig. 1) with a
PrP glycoform ratio that showed a dominant diglycosylated
conformer, typical of that associated withCWD prions (Race
et al., 2002). In contrast, identical analysis of brain
homogenates prepared from uninfected mule deer showed
no detectable PK-resistant PrP (Fig. 1 and data not shown).
CWD prions do not transmit prion disease to
transgenic mice overexpressing human prion
PrPSc-positive CWD-infected brain and spinal cord homo-
genates were used to prepare inocula for transmission
studies in transgenic mice overexpressing human PrP with
M. K. Sandberg and others
2652 Journal of General Virology 91
either methionine or valine at polymorphic residue 129.
129MM Tg35, 129MM Tg45 and 129VV Tg152 transgenic
mice overexpress human PrP in brain at levels of two, four
and six times that of human brain, respectively (Collinge
et al., 1995b, 1996; Hill et al., 1997; Asante et al., 2002).
These lines of mice have been extensively used by us for over
15 years and have proven susceptibility to infection with
humanorBSEprions(Collingeet al.,1995b,1996; Hill etal.,
1997; Asante et al., 2002, 2006; Wadsworth et al., 2004, 2007,
2008a). Following intracerebral inoculation with CWD
brain or spinal cord, groups of 10 transgenic mice were
observedthroughout their lifetime for clinicalsigns of prion
disease.As reported in Table 1,weobserved noclinicalprion
disease in any inoculated mouse, including those with post-
inoculation intervals greater than 700 days (Table 1).
Accordingly, brains from mice culled as a result of inter-
current illness or senescence were examined for subclinical
prion transmission. In all cases examined, pathological PrP
accumulation in brain was undetectable by either immuno-
blotting (Fig. 2, Table 1) or immunohistochemistry (Fig. 3,
Table 1). Futhermore, neuropathological examination of
CWD-inoculated transgenic mouse brain, showed no
evidence of spongiform change or gliosis consistent with
prion disease and their appearance was indistinguishable
from the brain of age matched control mice inoculated with
normal mule deer brain (Fig. 3 and data not shown). In
summary, we conclude that intracerebral challenge of these
transgenic mice with CWD prions caused no clinical or
subclinical prion infection, indicating that both methionine
and valine 129 polymorphs of human PrP are refractory to
pathological conversion by CWD prions.
In this study, we have shown that transgenic mice over-
expressing human PrP of both residue 129 polymorphic
forms, known to be susceptible to a wide range of human
and other prions, are highly resistant to infection with
mule deer CWD prions. These findings agree with those of
others who have previously reported an inability of CWD
prions to transmit disease to transgenic mice expressing
human PrP 129 methionine (Kong et al., 2005; Tamguney
et al., 2006) or a poor ability of human PrP to act as a
substrate for CWD prions in in vitro conversion assays
(Raymond et al., 2000; Kurt et al., 2009). Importantly, the
transgenic mice used in our study have proven suscept-
ibility to infection with BSE prions [Hill et al., 1997; Asante
et al., 2002, 2006; Wadsworth et al., 2004 (Table 1)]. The
negative transmissions that we report here therefore
strongly support the conclusion that the transmission
barrier associated with the interaction of human PrP and
these CWD prions is greater than that associated with
interaction of human PrP and the prion strain causing
epizootic BSE in cattle.
The failure to show propagation of CWD prions using
human PrP as a substrate either in vivo in transgenic mice
or in vitro in biochemical conversion assays suggests that
potential zoonotic threat from CWD is low. However, an
important caveat in this regard is that the number of
prion strains propagated in CWD is currently unknown
Fig. 1. Detection of PrPScin the brain and spinal cord from CWD-
infected mule deer. Immunoblots show the analysis of 5 ml aliquots
of 10% (w/v) homogenates of uninfected mule deer brain or
CWD-infected mule deer brain or spinal cord, before (”) or after
(+) digestion with PK. Immunoblots were analysed by enhanced
chemiluminescence with anti-PrP monoclonal antibody ICSM35.
Table 1. Primary transmission of CWD and BSE prions to
Data for BSE transmissions have been published previously (Hill
et al., 1997; Asante et al., 2002).
CWD brain CWD spinal
*All mice were inoculated with 30 ml of 1% (w/v) tissue homogenate.
Attack rate is defined as the total number of both clinically affected
and subclinically infected mice as a proportion of the number of
inoculated mice. Subclinical prion infection was assessed by sodium
phosphotungstic acid precipitation of 250 ml 10% brain homogenate
and analysis for PrPScby immunoblotting and/or immunohisto-
chemical examination of brain.
DMice culled at 274, 316, 321, 436, 517, 517, 587 and 781 days post-
dMice culled at 354, 364, 463, 541, 704 and 724 days post-
§Mice culled at 322, 322, 395, 400, 529, 656 and 736 days post-
||Mice culled at 275, 345, 396, 462, 462 and 532 days post-
Mice culled at 341, 559, 662, 662, 680, 707, 707, 747 and 748 days
#Mice culled at 392, 414, 542, 699 and 732 days post-inoculation.
CWD transmission studies in human PrP transgenic mice
(Browning et al., 2004; Raymond et al., 2007; Green et al.,
2008; Angers et al., 2010). Because prion strains can adapt
and mutate on passage in new species (Collinge & Clarke,
2007; Beringue et al., 2008; Castilla et al., 2008; Collinge,
2010), and also within species as a result of PrP
polymorphisms and other genetic factors (Asante et al.,
2002; Lloyd et al., 2004; Wadsworth et al., 2004; Mead et
al., 2009; Lloyd et al., 2009), the risk that each prion strain
poses to public health must be evaluated directly. There is
now growing evidence that polymorphisms of cervid PrP
may dictate prion strain selection (O’Rourke et al., 2004;
Meade-White et al., 2007; Green et al., 2008; Angers et al.,
2010). Thus, while the available experimental data appear
reassuring, further transmission studies will be of vital
importance to evaluate the properties of distinct cervid
prion strains as they are isolated.
Mule deer tissues. Importation, storage and use of CWD-infected
tissues was performed under licence granted by Defra under the terms
of the Importation of Animal Pathogens Order 1980. CWD-infected
mule deer brain (from animal D10) and spinal cord (from animal D08)
that had clinical signs consistent with terminal stages of prion disease.
CWD-infection in these animals was confirmed by the presence of
histopathological lesions in the brain, including spongiform degenera-
tion of the perikaryon, by immunohistochemical or immunoblot
detection of disease-related PrP and by positive transmission of prion
disease to transgenic mice expressing cervid PrP (Browning et al., 2004;
Angersetal., 2006;Green etal., 2008). Brain fromuninfectedmule deer
fawns (FPS 6.98 and FPS 3.98) was used as negative controls.
Transgenic mice. Transgenic mice homozygous fora human PrP 129V
transgene array and murine PrP null alleles (Prnpo/o) designated
Tg(HuPrP129V+/+Prnpo/o)-152 mice (129VV Tg152 mice) or homo-
(Prnpo/o) designated Tg(HuPrP129M+/+Prnpo/o)-35 mice (129MM
Tg35 mice) or Tg(HuPrP129M+/+Prnpo/o)-45 mice (129MM Tg45
mice) have been described previously (Collinge et al., 1995b, 1996; Hill
Fig. 2. Failure to detect PrPScin the brain of CWD prion-
inoculated transgenic mice. The high sensitivity immunoblot using
anti-PrP monoclonal antibody 3F4 shows PK-digested sodium
phosphotungstic acid pellets recovered from 10% (w/v) trans-
genic mouse brain homogenates. Lanes 1 and 2, positive controls
showing efficient recovery of PrPScafter spiking 2 ml 10% (w/v)
BSE-inoculated 129MM Tg45 and 129MM Tg35 transgenic
mouse brain homogenates (Asante et al., 2002) into 100 ml
10% (w/v) uninfected 129MM Tg45 and 129MM Tg35 mouse
brain homogenates, respectively. Lane 3, PK-digested sodium
phosphotungstic acid pellet from 250 ml 10% (w/v) brain
homogenate from a 129MM Tg45 mouse inoculated with normal
mule deer brain. Lanes 4–9, PK-digested sodium phosphotungstic
acid pellets from 250 ml 10% (w/v) brain homogenates from
129MM Tg35, 129MM Tg45 and 129VV Tg152 mice inoculated
with CWD-infected mule deer brain.
Fig. 3. Failure to detect abnormal PrP deposition in the brain of CWD prion-inoculated transgenic mice. Representative PrP
immunohistochemistry using anti-PrP monoclonal antibody ICSM35. Panels (a–f) show no abnormal PrP deposition in either the
hippocampus or thalamus of 129VV Tg152, 129MM Tg45 or 129MM Tg35 mice inoculated with CWD-infected brain
homogenate. These mice were culled 517, 529 and 559 days post-inoculation, respectively. Panels (g) and (h) show
hippocampus and thalamus from an age matched control 129MM Tg45 mouse brain inoculated with 10% (w/v) uninfected
mule deer brain homogenate. In contrast, extensive deposition of abnormal PrP is seen in the hippocampus and thalamus of a
BSE-infected 129MM Tg45 mouse with subclinical prion disease (panels i and j) (Asante et al., 2002). Bar, 500 mm.
M. K. Sandberg and others
2654 Journal of General Virology 91
Transmission studies. All procedures were carried out in a
microbiological containment level 3 facility with strict adherence to
safety protocols. Care of mice was according to institutional
guidelines. Mule deer tissues were prepared as 10% (w/v) homo-
genates in sterile PBS lacking Ca2+and Mg2+ions by serial passage
through needles of decreasing diameter, and subsequently diluted to
1% (w/v) in PBS. Following intracerebral inoculation with 30 ml of
1% (w/v) tissue homogenate as described previously (Asante et al.,
2002, 2006; Wadsworth et al., 2004), mice were examined daily and
were killed if exhibiting signs of distress or once a diagnosis of clinical
prion disease was established. Brains from inoculated mice were
analysed by PrP immunoblotting or immunohistochemistry and by
Immunoblotting. Allprocedureswere carriedout ina microbiological
containment level 3 facility with strict adherence to safety protocols.
Tissue homogenates (10% w/v) were prepared in PBS lacking Ca2+or
ions. PK digestion (50 or 100 mg ml21
concentration, 1 h, 37 uC), electrophoresis and immunoblotting was
performed as described previously (Wadsworth et al., 2001, 2008b).
Immunoblot detection was performed using anti-PrP monoclonal
antibody ICSM35 (D-Gen) for cervid PrP or 3F4 (Kascsak et al., 1987)
for human PrP in transgenic mice. Brain homogenates scored negative
for PrPScafter analysis of 10 ml 10% (w/v) brain homogenate were re-
analysed by sodium phosphotungstic acid precipitation of PrPSc(Safar
et al., 1998) from 250 ml of 10% (w/v) brain homogenate as described
previously (Wadsworth et al., 2001).
Neuropathology and immunohistochemistry. All steps prior to
prion decontamination with formic acid were performed within a
microbiological containment level 3 facility with strict adherence to
safety protocols. Brain was fixed in 10% buffered formal saline and
then immersed in 98% formic acid for 1 h and paraffin wax
embedded. Serial sections of 4 mm thickness were pre-treated by
boiling for 10 min in a low ionic strength buffer (2.1 mM Tris,
1.3 mM EDTA, 1.1 mM sodium citrate, pH 7.8) before exposure to
98% formic acid for 5 min. Abnormal PrP accumulation was
examined using anti-PrP monoclonal antibody ICSM35 (D-Gen) on
a Ventana automated immunohistochemical staining machine
(Ventana Medical Systems) using proprietary secondary detection
reagents (Ventana Medical Systems) before development with 393-
diaminobenzedine tetrachloride as the chromogen (Wadsworth et al.,
2008b). Harris haematoxylin and eosin staining was done by
conventional methods. Appropriate positive and negative controls
were used throughout. Photographs were taken on an ImageView
digital camera and composed with Adobe Photoshop.
We thank our biological service team for animal care and R. Young
for the preparation of figures. We thank Michael W. Miller and the
Colorado Division of Wildlife for access to the CWD-infected deer
samples. This research was funded by the Medical Research Council
(UK) and the European Union. Conflict of interest statement: J.C. is
a Director and J.C. and J.D.F.W. are shareholders and consultants of
D-Gen Limited, an academic spin-out company working in the field
of prion disease diagnosis, decontamination and therapeutics. D-Gen
markets the ICSM35 antibody used in this study.
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