Prion infectivity in the spleen of a PRNP heterozygous individual with subclinical variant Creutzfeldt–Jakob disease

Article (PDF Available)inBrain 136(4) · February 2013with47 Reads
DOI: 10.1093/brain/awt032 · Source: PubMed
Abstract
Blood transfusion has been identified as a source of human-to-human transmission of variant Creutzfeldt-Jakob disease. Three cases of variant Creutzfeldt-Jakob disease have been identified following red cell transfusions from donors who subsequently developed variant Creutzfeldt-Jakob disease and an asymptomatic red cell transfusion recipient, who did not die of variant Creutzfeldt-Jakob disease, has been identified with prion protein deposition in the spleen and a lymph node, but not the brain. This individual was heterozygous (MV) at codon 129 of the prion protein gene (PRNP), whereas all previous definite and probable cases of variant Creutzfeldt-Jakob disease have been methionine homozygotes (MM). A critical question for public health is whether the prion protein deposition reported in peripheral tissues from this MV individual correlates with infectivity. Additionally it is important to establish whether the PRNP codon 129 genotype has influenced the transmission characteristics of the infectious agent. Brain and spleen from the MV blood recipient were inoculated into murine strains that have consistently demonstrated transmission of the variant Creutzfeldt-Jakob disease agent. Mice were assessed for clinical and pathological signs of disease and transmission data were compared with other transmission studies in variant Creutzfeldt-Jakob disease, including those on the spleen and brain of the donor to the index case. Transmission of variant Creutzfeldt-Jakob disease was observed from the MV blood recipient spleen, but not from the brain, whereas there was transmission from both spleen and brain tissues from the red blood cell donor. Longer incubation times were observed for the blood donor spleen inoculum compared with the blood donor brain inoculum, suggesting lower titres of infectivity in the spleen. The distribution of vacuolar pathology and abnormal prion protein in infected mice were similar following inoculation with both donor and recipient spleen homogenates, providing initial evidence of similar transmission properties after propagation in PRNP codon 129 MV and MM individuals. These studies demonstrate that spleen tissue from a PRNP MV genotype individual can propagate the variant Creutzfeldt-Jakob disease agent and that the infectious agent can be present in the spleen without CNS involvement.
BRAIN
A JOURNAL OF NEUROLOGY
Prion infectivity in the spleen of a
PRNP
heterozygous individual with subclinical
variant Creutzfeldt–Jakob disease
Matthew T. Bishop,
1,
* Abigail B. Diack,
2,
* Diane L. Ritchie,
1
James W. Ironside,
1
Robert G. Will
1,
and Jean C. Manson
2,
1 National Creutzfeldt–Jakob Disease Research and Surveillance Unit, University of Edinburgh, Bryan Matthews Building, Western General Hospital,
Crewe Road, Edinburgh, EH4 2XU, UK
2 Neurobiology Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian,
EH25 9RG, UK
These authors contributed equally to this work.
Correspondence to: Professor Jean Manson,
Head of Neurobiology Division,
The Roslin Institute and Royal (Dick) School of Veterinary Studies,
University of Edinburgh,
Easter Bush, Midlothian,
EH25 9RG, UK
E-mail: jean.manson@roslin.ed.ac.uk
Blood transfusion has been identified as a source of human-to-human transmission of variant Creutzfeldt–Jakob disease. Three
cases of variant Creutzfeldt–Jakob disease have been identified following red cell transfusions from donors who subsequently
developed variant Creutzfeldt–Jakob disease and an asymptomatic red cell transfusion recipient, who did not die of variant
Creutzfeldt–Jakob disease, has been identified with prion protein deposition in the spleen and a lymph node, but not the brain.
This individual was heterozygous (MV) at codon 129 of the prion protein gene (
PRNP
), whereas all previous definite and
probable cases of variant Creutzfeldt–Jakob disease have been methionine homozygotes (MM). A critical question for public
health is whether the prion protein deposition reported in peripheral tissues from this MV individual correlates with infectivity.
Additionally it is important to establish whether the
PRNP
codon 129 genotype has influenced the transmission characteristics of
the infectious agent. Brain and spleen from the MV blood recipient were inoculated into murine strains that have consistently
demonstrated transmission of the variant Creutzfeldt–Jakob disease agent. Mice were assessed for clinical and pathological
signs of disease and transmission data were compared with other transmission studies in variant Creutzfeldt–Jakob disease,
including those on the spleen and brain of the donor to the index case. Transmission of variant Creutzfeldt–Jakob disease was
observed from the MV blood recipient spleen, but not from the brain, whereas there was transmission from both spleen and
brain tissues from the red blood cell donor. Longer incubation times were observed for the blood donor spleen inoculum
compared with the blood donor brain inoculum, suggesting lower titres of infectivity in the spleen. The distribution of vacuolar
pathology and abnormal prion protein in infected mice were similar following inoculation with both donor and recipient
spleen homogenates, providing initial evidence of similar transmission properties after propagation in
PRNP
codon 129 MV
and MM individuals. These studies demonstrate that spleen tissue from a
PRNP
MV genotype individual can propagate
the variant Creutzfeldt–Jakob disease agent and that the infectious agent can be present in the spleen without CNS
involvement.
doi:10.1093/brain/awt032 Brain 2013: 136; 1139–1145 | 1139
Received August 13, 2012. Revised January 10, 2013. Accepted January 14, 2013. Advance Access publication February 28, 2013
ß The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/),
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: variant Creutzfeldt–Jakob disease; infection; subclinical; blood transfusion; prion
Abbreviations: HuMM = transgenic mouse line expressing human prion protein with MM genotype at codon 129; PrP
Sc
= disease
associated (‘scrapie’) form of the prion protein
Introduction
Creutzfeldt–Jakob disease is a rare human prion disease that
occurs sporadically, in association with inherited gene mutations
or may be acquired (Prusiner, 1998). A novel form of Creutzfeldt–
Jakob disease, variant Creutzfeldt–Jakob disease, was first reported
in the UK in 1996 (Will et al., 1996) and since then 176 UK
patients and 51 patients from 11 other countries (www.cjd.ed.
ac.uk) have been identified. The hypothesis that variant
Creutzfeldt–Jakob disease is caused by infection with bovine
spongiform encephalopathy has been supported by a range of
evidence, including experimental animal studies demonstrating
close similarities between the agent strain of variant Creutzfeldt–
Jakob disease and bovine spongiform encephalopathy (Bruce
et al., 1997; Hill et al., 1997). Surveillance systems are in place
in many countries to monitor both bovine spongiform encephal-
opathy and variant Creutzfeldt–Jakob disease and, while the
number of cases of bovine spongiform encephalopathy worldwide
is clearly in decline, there is still considerable uncertainty surround-
ing the future course of the variant Creutzfeldt–Jakob disease out-
breaks in the UK and other countries.
The codon 129 polymorphism in the prion protein gene (PRNP)
is known to influence susceptibility to Creutzfeldt–Jakob disease
(Pocchiari et al., 2004). All definite and probable cases of variant
Creutzfeldt–Jakob disease with data on genotype have occurred
in individuals homozygous for methionine at codon 129 (MM129).
Experimental transmission studies indicate that individuals of
all codon 129 genotypes may be susceptible to variant
Creutzfeldt–Jakob disease infection (Bishop et al., 2006). The in-
cubation period in individuals with the MV or VV genotypes is
likely to be longer than those with the MM genotype and
these individuals may never develop clinical disease. This is of
major significance to public health as subclinical infection has
the potential to increase the risks of onward secondary transmis-
sion of infection from person to person, for example by blood
transfusion.
The number of individuals with asymptomatic variant
Creutzfeldt–Jakob disease infection in the UK population is un-
known, with estimates ranging from 900 (statistical modelling;
Clarke et al., 2007) to 3800 (screening of anonymous appendix
and tonsil tissue archives; Hilton et al., 2004). One recent model-
ling study, which includes the potential for all codon 129 genotype
individuals to be at risk and the possibility of unidentifiable blood
transfusion infections, suggests that there will be between 84 and
3000 cases of variant Creutzfeldt–Jakob disease in the years 2010
to 2179 (Garske and Ghani, 2010). The most recent appendix
tissue archive survey has indicated a prevalence of asymptomatic
variant Creutzfeldt–Jakob disease infection of around 1 in 2000 in
the UK population (http://www.hpa.org.uk/hpr/archives/2012/
hpr3212.pdf) (HPA, 2012).
The identification in 2004 of the first case of transfusion
associated variant Creutzfeldt–Jakob disease infection (Llewelyn
et al., 2004), through non-leucodepleted red cells, provided evi-
dence of human-to-human transmission of variant Creutzfeldt–
Jakob disease. Two additional cases of transfusion transmission
of variant Creutzfeldt–Jakob disease have since been identified in
MM individuals (Wroe et al., 2006). In addition a patient, who
died of non-Creutzfeldt–Jakob disease related causes, has been
shown to have abnormal prion protein deposition in the spleen
and a lymph node, but not the brain (Peden et al., 2004). This
individual, with an MV genotype, had received a red blood cell
transfusion from an MM donor who subsequently died of variant
Creutzfeldt–Jakob disease. This is the first direct evidence demon-
strating that variant Creutzfeldt–Jakob disease infection can occur
in an individual with an MV genetic background.
This is of significant potential importance for public health as
50% of Caucasian populations are codon 129 heterozygotes and
carrier states of prion infection would increase the risk of second-
ary transmission of infection. It is therefore of critical importance
to determine whether deposition of the disease associated (‘scra-
pie’) form of the prion protein (PrP
Sc
) in the MV spleen is asso-
ciated with actual prion infectivity (Wadsworth et al., 2011).
Furthermore, there is the potential for the properties of the infec-
tious agent to change after secondary transmission within a spe-
cies or in a different genetic background and it is therefore
important to determine whether the disease agent characteris-
tics in the spleen of the MV individual are distinct from that of
primary variant Creutzfeldt–Jakob disease infection in an MM
background.
Materials and methods
Mouse lines
Transgenic mice expressing human prion protein (HuMM) have been
developed by the Neurobiology Division (The Roslin Institute) by a
methodology that is detailed elsewhere (Bishop et al., 2006). These
inbred mice are homozygous for methionine at codon 129 and express
human prion protein under the control of the original mouse prion
gene regulatory region. Wild-type RIII (also referred to as MR)
mouse lines are used routinely at The Roslin Institute for transmis-
sible spongiform encephalopathy transmission studies. They have pre-
viously been inoculated with a number of isolates from variant
Creutzfeldt–Jakob disease and develop clinical disease and pathology
at 300–450 days post injection dependent on the inoculation mater-
ial and method (Bruce et al., 2004; Ritchie et al., 2009; Diack et al.,
2012).
Inoculation
The variant Creutzfeldt–Jakob disease brain and spleen tissue samples
were supplied by the tissue bank in the National Creutzfeldt–Jakob
1140 | Brain 2013: 136; 1139–1145 M. T. Bishop et al.
Disease Research and Surveillance Unit, part of the Edinburgh Brain
Bank funded by the MRC (G1100616). Consent was given for use of
these tissues in research, and the tissue bank has ethics approval
(Lothian NHS Board, Research Ethics Committee, Reference: 11/ES/
0022). Transgenic mice were anaesthetized with halothane and
injected with 0.02 ml of 1% w/v brain or spleen homogenate into
the right cerebral hemisphere. Wild-type mice received a 0.02 ml
dose of 10% w/v homogenate and an additional intraperitoneal injec-
tion of 0.1 ml. From 100 days the mice were scored for signs of
disease (Fraser and Dickinson, 1968). Mice were sacrificed by cervical
dislocation at the terminal stage of disease or for welfare reasons.
The brains of the mice were recovered at post-mortem and cut sagit-
tally, with one half snap-frozen in liquid nitrogen for biochemical ana-
lysis and the remaining half fixed for histology. The animal
experiments were approved by The Roslin Institute’s ethical review
committee and they were conducted according to the regulations of
the United Kingdom Home Office Animals (Scientific Procedures)
Act 1986.
Prion protein detection by
immunocytochemistry
Following fixation in 10% formal saline for 48 h, the mouse half-brains
were treated for 1.5 h in 98% formic acid, cut transversely into four
sections, and embedded in paraffin. Sections of 6 -mm thickness were
cut for immunocytochemistry. Prion protein was detected by use of
the Vectastain Elite ABC Kit (Vector Labs) with overnight anti-prion
protein primary antibody incubation (6H4, Prionics, 1:5000). Detection
of antibody binding was through deposition of 3,3’-diaminobenzidine
chromogen.
Scoring of vacuolation
Haematoxylin and eosin stained brain sections from each mouse were
scored blind for degree of vacuolation (ranked 0 to 5) found in nine
grey matter regions and three white matter regions (Fraser and
Dickinson, 1968). The brain regions analysed were: grey matter
areas: (GM1) dorsal medulla, (GM2) cerebellar cortex, (GM3) superior
colliculus, (GM4) hypothalamus, (GM5) thalamus, (GM6) hippocam-
pus, (GM7) septum, (GM8) retrosplenial and adjacent motor cortex,
(GM9) cingulate and adjacent motor cortex; white matter areas:
(WM1) cerebellar white matter, (WM2) mesencephalic tegmentum,
(WM3) pyramidal tract. Mean scores were calculated for the RIII
mouse groups to produce lesion profiles.
Western blot analysis
Frozen brain samples were homogenized using a micro-pestle in nine
volumes of Tris-buffered saline (pH 7.6), containing 0.5% Nonidet
TM
P40 and 0.5% sodium deoxycholate, to give a 10% w/v suspension.
This material was cleared by centrifugation at 420 g for 5 min at 4
C
and the supernatant treated with 50 mg/ml proteinase K for 1 h at
37
C. For further details see Head et al., (2004). For samples with
low levels of PrP
Sc
the proteinase K treated material (0.1 ml) was
centrifuged at 14 000 rpm for 1 h at 4
C, the supernatant discarded,
then the pellet resuspended in 0.02 ml loading buffer. The digested
product was heat denatured for 10 min at 100
C then loaded onto a
10% Bis/Tris NuPAGE
Õ
Novex gel (Invitrogen). After electrophoresis
the gel was blotted onto polyvinylidene difluoride membrane.
Detection of prion protein used the ECL + technique (Amersham
Biosciences) with primary antibody 6H4 (Prionics) at 1:40 000 and
an anti-mouse IgG peroxidase-linked secondary antibody (Amersham
Biosciences) at 1:40 000. Images were captured on X-ray film and
using a Bio-Rad ChemiDoc XRS + imaging system. Western blot ana-
lysis of proteinase K-treated PrP
Sc
, detected with the 6H4 antibody,
typically results in three bands of unglycosylated, monoglycosylated
and diglycosylated fragments with the highest molecular weight digly-
cosylated band running at 30 kDa and lowest molecular weight
unglycosylated band running at 21 kDa (termed type 1) or 19 kDa
(termed type 2) (Head et al., 2004).
Results
Inoculation of brain tissue homogenate
from MM blood donor and MV blood
recipient
Inoculation of brain tissue homogenate from the variant
Creutzfeldt–Jakob disease blood donor resulted in clinical disease
and pathology in both HuMM and RIII mouse lines (Table 1,
Figs 1 and 2). Incubation times were similar to those previously
observed in these mouse lines following inoculation of brain tissue
homogenate from other patients with variant Creutzfeldt–Jakob
disease (Bruce et al., 2001; Bishop et al., 2006; Ritchie et al.,
2009; Diack et al., 2012).
In contrast, there was no clinical or pathological evidence for
transmission of disease from inoculum derived from the MV blood
recipient brain tissue in either HuMM or RIII mice.
Immunohistochemical analysis of the HuMM and RIII mice inocu-
lated with the blood donor brain homogenate showed a variability
in amount of PrP
Sc
but similar distribution pattern of PrP
Sc
in the
brain, to previously published variant Creutzfeldt–Jakob disease
transmission studies (Bishop et al., 2006; Ritchie et al., 2009;
Diack et al., 2012).
Western blot analysis of PrP
Sc
extracted from the brains of both
HuMM and RIII mice inoculated with the variant Creutzfeldt–
Jakob disease blood donor brain material showed a type 2 mobility
(of the lower unglycosylated fragment) as expected for variant
Creutzfeldt–Jakob disease transmissions to these mice (Figs 3
and 4). PrP
Sc
was not detectable in mice inoculated with the
MV blood recipient brain tissue (HuMM data shown in Fig. 3,
Lane 4).
Thus, there was no evidence of transmissible variant
Creutzfeldt–Jakob disease infection in the sample of brain tissue
from the MV genotype blood transfusion recipient, while the brain
tissue homogenate from the variant Creutzfeldt–Jakob disease
blood donor has similar transmission properties to previously re-
ported cases with variant Creutzfeldt–Jakob disease (Bruce et al.,
2001; Bishop et al., 2006; Ritchie et al., 2009; Diack et al., 2012).
This lack of transmission adds further evidence to the hypothesis
that the variant Creutzfeldt–Jakob disease infection had not
reached the CNS as initially proposed by the absence of variant
Creutzfeldt–Jakob disease pathology in the brain of this patient
(Peden et al., 2004).
Prion infectivity in subclinical vCJD Brain 2013: 136; 1139–1145 | 1141
Table 1 Primary inoculation into HuMM transgenic and RIII wild-type mouse lines
Inoculum source HuMM RIII
Clinical positive and incubation period
MM blood donor Brain 2/17 (529 days) 18/28 (404 days)
MV blood recipient Brain 0/18 0/21
MM blood donor Spleen 0/18 5/21 (680 days)
MV blood recipient Spleen 1/18 (611 days) 12/23 (609 days)
Vacuolation-positive
MM blood donor Brain 7/17 21/28
MV blood recipient Brain 0/18 0/21
MM blood donor Spleen 3/18 3/21
MV blood recipient Spleen 1/18 11/22
Immunocytochemistry- positive for PrP
Sc
MM blood donor Brain 16/16 25/28
MV blood recipient Brain 0/18 0/21
MM blood donor Spleen 16/17 7/20
MV blood recipient Spleen 9/19 14/23
Figure 1 Comparison of immunocytochemistry data from HuMM and RIII mice inoculated with brain and spleen derived inoculum from
the MM variant Creutzfeldt–Jakob (vCJD) disease blood donor, and spleen derived inoculum from the MV blood recipient. Scale
bar = 200 mm; anti-prion protein antibody 6H4; arrow = CA2 region.
Figure 2 Comparison of lesion profile data from RIII mice. Red line ( ) = MM blood donor brain (n=21); blue line ( + ) = MM blood
donor spleen (n=3); green line (O) = MV blood recipient spleen (n=11); brain regions are listed in the ‘Materials and methods’ section.
1142 | Brain 2013: 136; 1139–1145 M. T. Bishop et al.
Inoculation of spleen tissue
homogenate from the MM blood
donor and MV blood recipient showed
similar transmission properties
Clinical signs and/or transmissible spongiform encephalopathy
pathology were present in RIII and HuMM mice inoculated with
the MM blood donor spleen homogenate and the MV blood re-
cipient spleen homogenate (Table 1, Figs 1 and 2). A comparison
of incubation periods between MM blood donor spleen and brain
inocula suggested a lower infectivity titre in the spleen, as reported
previously (Ritchie et al., 2009).
A lower proportion of RIII mice were scored positive for clinical
and pathological signs with the MM blood donor spleen inoculum
in comparison to the MV blood recipient spleen, suggesting a
higher titre of infectivity in the spleen of the MV blood recipient.
However, only 1 of 18 of the HuMM transgenic mice inoculated
with the MV blood recipient spleen inoculum was positive for
transmissible spongiform encephalopathy vacuolation in the brain
whereas 3 of 18 HuMM mice inoculated with the MM blood
donor spleen inoculum were positive. One potential explanation
for these differences is variation in levels of infectivity resulting
from differences in tissue sampling. However, this is an unlikely
explanation since the transgenic and wild-type mice gave opposite
results. An alternative explanation is that the infectivity within the
spleen of the MV blood recipient replicated less efficiently in the
HuMM mice.
Lesion profiles for the RIII mice indicated that inoculation of
blood donor and recipient spleen tissue resulted in similar
pathological targeting in the brain (Fig. 2). This lesion profile is
similar to that observed in RIII mice after inoculation with variant
Creutzfeldt–Jakob disease infected brain material (Ritchie et al.,
2009). Lesion profiles are usually generated from a minimum of
five mice and, although data from only three mice inoculated with
the MM blood donor spleen tissue were available, the mean
scores closely follow the pattern seen for the MV blood recipient
spleen inoculum. The three characteristic peaks in vacuolation in-
tensity were for the brain regions: GM1 (dorsal medulla), GM4
(hypothalamus) and GM7 (septum).
Although there was a degree of variation between individual
mice, there were no major differences in either the distribution
(targeting) or morphology of the PrP
Sc
deposits in the brains of
HuMM and RIII mice inoculated with spleen homogenate from
the MM blood donor and MV recipient (Fig. 1). RIII mice ex-
hibited more widespread PrP
Sc
deposition than the HuMM, with
a granular pattern of deposition, and intense staining in the CA2
region of the hippocampus; the thalamus; the medulla; and the
pons. Plaque-like aggregates of PrP
Sc
in the molecular layer of the
CA1 region of the hippocampus and more diffuse staining in white
matter fibres were seen in HuMM mice. All these deposition pat-
terns have been seen before in previous studies and are charac-
teristic of the transmission properties of variant Creutzfeldt–Jakob
disease brain and lymphoid tissues (Fig. 1) (Brown et al., 2003;
Bishop et al., 2006; Ritchie et al., 2009).
PrP
Sc
deposited in the brains of the HuMM mice inoculated with
the blood donor brain and spleen tissue both gave a type 2 mo-
bility unglycosylated band pattern on western analysis (Fig. 3). In
HuMM mice, the amount of PrP
Sc
detected by immunocytochem-
istry analysis following inoculation of the MV blood recipient
spleen is low and extremely focal when compared with that
observed with the MM blood donor spleen inoculum (Fig. 1)
and as expected proved to be too low for detection by western
analysis even when using concentrating methods of preparation
(Fig. 3, Lane 5). Samples of brain tissue homogenate from HuMM
mice inoculated with the MV blood recipient brain tissue, that
were negative by immunocytochemistry, were also confirmed as
negative by this concentration method (Fig. 3, Lane 4). Western
analysis of the brains of the infected RIII mice demonstrated that
both MV blood recipient spleen tissue inoculation and MM blood
donor brain inoculation showed a similar type 2 mobility unglyco-
sylated band pattern (Fig. 4). Tissue samples for western analysis
were not available from RIII mice infected with the MM blood
donor spleen tissue and samples from RIII mice infected with
Figure 3 Western blot data from HuMM mice inoculated with
brain and spleen derived variant Creutzfeldt–Jakob disease
inocula. Duplicate blots (A and B) exposed for different lengths
of time to visualize band mobility. Lanes: (1) sporadic
Creutzfeldt–Jakob disease type 1; (2) blood donor brain tissue
inoculum; (3) blood donor spleen tissue inoculum; (4) blood
recipient brain tissue inoculum; (5) blood recipient spleen tissue
inoculum; (6) variant Creutzfeldt–Jakob disease type 2B.
Molecular weight marker position shown at 20 kDa and 30 kDa.
Antibody = 6H4.
Figure 4 Western blot data from RIII mice inoculated with brain
and spleen derived variant Creutzfeldt–Jakob disease inocula.
Lanes: (1) molecular weight marker (20 kDa and 30 kDa); (2)
blood donor brain tissue inoculum; (3) blood recipient spleen
tissue inoculum. Antibody = 6H4.
Prion infectivity in subclinical vCJD Brain 2013: 136; 1139–1145 | 1143
the MV blood recipient brain tissue were negative by the concen-
tration method.
Discussion
This study provides definitive evidence that spleen tissue from an
asymptomatic individual contains variant Creutzfeldt–Jakob disease
infectivity and that the variant Creutzfeldt–Jakob disease agent
retains infectivity following passage through an MV genotype
host. The findings are of importance as there has been uncertainty
as to whether prion protein immunostaining in peripheral tissues
from non-clinical variant Creutzfeldt–Jakob disease necessarily cor-
related with infectivity (Hilton et al., 2004; Wadsworth et al.,
2011). The demonstration of infectivity in such tissues underlines
the potential for asymptomatic carriers of variant Creutzfeldt–
Jakob disease infection to pose a risk of secondary transmission
of infection through blood transfusion or contamination of surgical
instruments. The incubation times recorded in this study would
suggest moderate but significant levels of infectivity are present
in the spleen of the MV genotype recipient, raising the possibility
that other peripheral tissues and the blood of this individual are
also infected, as indicated by immunohistochemistry in the initial
report of this case (Peden et al., 2004). Furthermore the confirm-
ation that there is prion infectivity in an individual with a PRNP
codon 129 MV genotype indicates that this genetic subgroup,
which accounts for 50% of the UK population, can act as carriers
of variant Creutzfeldt–Jakob disease infection.
Our results also provide initial evidence that the variant
Creutzfeldt–Jakob disease transmission properties in the MV
blood recipient spleen tissue are similar to those of the MM
blood donor. This is a critical issue for public health as there is a
potential for these infectious agents to change characteristics,
including virulence, after serial transmission or following passage
in a different genetic background. The relative stability of the
agent also makes it more likely that the clinical phenotype of
variant Creutzfeldt–Jakob disease infection in an MV background
may be similar to that of the well-recognized clinical phenotype in
an MM background. While a first passage of an agent between
species is not normally sufficient to confirm strain identity, the
similarities identified at first passage in this study are striking.
We are now undertaking a more extensive strain comparison by
our standard serial-passage method using inocula derived from this
primary transmission experiment (Ritchie et al., 2009). This will
help establish whether the strain characteristics have indeed re-
mained stable following passage through an MV host.
This study has established that variant Creutzfeldt–Jakob disease
infectivity can be replicated within a PRNP codon 129 MV geno-
type host and within a non-CNS tissue, in the absence of variant
Creutzfeldt–Jakob disease pathology in the brain. This demon-
strates the potential for asymptomatic carriage of variant
Creutzfeldt–Jakob disease infection in the UK population, under-
lining the risk of a silent subclinical epidemic that could result from
transfer of infection through blood transfusion or surgery (Garske
and Ghani, 2010). It is imperative therefore that continued active
surveillance and infection control measures for variant Creutzfeldt–
Jakob disease are continued into the future.
Acknowledgements
We gratefully acknowledge the assistance of the Biological
Resource Facility staff for clinical assessment of the mice and the
Pathology Division for sectioning the mouse brains and assessing
the levels of transmissible spongiform encephalopathy vacuolation.
Mark Head, Laura McCulloch and Allister Smith gave assistance
with the analysis of western blots. We also wish to thank Enrico
Cancellotti for many discussions throughout this work.
Funding
This is an independent report commissioned and funded by the
Policy Research Programme in the Department of Health, United
Kingdom. The views expressed in the publication are those of the
authors and not necessarily those of the Department of Health.
Work undertaken at The Roslin Institute was also funded by the
Medical Research Council. The tissue bank in the National CJD
Research & Surveillance Unit is supported by the Medical
Research Council (G1100616).
References
Bishop MT, Hart P, Aitchison L, Baybutt HN, Plinston C, Thomson V,
et al. Predicting susceptibility and incubation time of human-to-human
transmission of vCJD. Lancet Neurol 2006; 5: 393–8.
Brown DA, Bruce ME, Fraser JR. Comparison of the neuropathological
characteristics of bovine spongiform encephalopathy (BSE) and variant
Creutzfeldt-Jakob disease (vCJD) in mice. Neuropathol Appl Neurobiol
2003; 29: 262–72.
Bruce ME, Boyle A, McConnel I. TSE strain typing in mice. In: Lehmann
S, Grassi J, editors. Techniques in prion research. Birkhauser Verlag;
2004 p. 132–46.
Bruce ME, McConnell I, Will RG, Ironside JW. Detection of variant
Creutzfeldt-Jakob disease infectivity in extraneural tissues. Lancet
2001; 358: 208–9.
Bruce ME, Will RG, Ironside JW, McConnell I, Drummond D, Suttie A,
et al. Transmissions to mice indicate that ‘new variant’ CJD is caused
by the BSE agent. Nature 1997; 389: 498–501.
Clarke P, Will RG, Ghani AC. Is there the potential for an epidemic of
variant Creutzfeldt-Jakob disease via blood transfusion in the UK? J R
Soc Interface 2007; 4: 675–84.
Diack AB, Ritchie D, Bishop M, Pinion V, Brandel JP, Haik S, et al.
Constant transmission properties of variant Creutzfeldt-Jakob disease
in 5 countries. Emerg Infect Dis 2012; 18: 1574–9.
Fraser H, Dickinson AG. The sequential development of the brain lesion
of scrapie in three strains of mice. J Comp Pathol 1968; 78: 301–11.
Garske T, Ghani AC. Uncertainty in the tail of the variant
Creutzfeldt-Jakob disease epidemic in the UK. PLoS One 2010; 5:
e15626.
Head MW, Bunn TJ, Bishop MT, McLoughlin V, Lowrie S, McKimmie CS,
et al. Prion protein heterogeneity in sporadic but not variant
Creutzfeldt-Jakob disease: UK cases 1991–2002. Ann Neurol 2004;
55: 851–9.
Hill AF, Desbruslais M, Joiner S, Sidle KC, Gowland I, Collinge J, et al.
The same prion strain causes vCJD and BSE. Nature 1997; 389:
448–50.
Hilton DA, Ghani AC, Conyers L, Edwards P, McCardle L, Ritchie D,
et al. Prevalence of lymphoreticular prion protein accumulation in
UK tissue samples. J Pathol 2004; 203: 733–9.
1144 | Brain 2013: 136; 1139–1145 M. T. Bishop et al.
HPA. Summary results of the second national survey of abnormal prion
prevalence in archived appendix specimens. Health Prot Rep 2012; 6.
Llewelyn CA, Hewitt PE, Knight RS, Amar K, Cousens S, Mackenzie J,
et al. Possible transmission of variant Creutzfeldt-Jakob disease by
blood transfusion. Lancet 2004; 363: 417–21.
Peden AH, Head MW, Ritchie DL, Bell JE, Ironside JW. Preclinical vCJD
after blood transfusion in a PRNP codon 129 heterozygous patient.
Lancet 2004; 364: 527–9.
Pocchiari M, Puopolo M, Croes EA, Budka H, Gelpi E, Collins S, et al.
Predictors of survival in sporadic Creutzfeldt-Jakob disease and other
human transmissible spongiform encephalopathies. Brain 2004; 127:
2348–59.
Prusiner SB. Prions. Proc Natl Acad Sci USA 1998; 95: 13363–83.
Ritchie DL, Boyle A, McConnell I, Head MW, Ironside JW, Bruce ME.
Transmissions of variant Creutzfeldt-Jakob disease from brain and
lymphoreticular tissue show uniform and conserved bovine spongiform
encephalopathy-related phenotypic properties on primary and second-
ary passage in wild-type mice. J Gen Virol 2009; 90 (Pt 12): 3075–82.
Wadsworth JD, Dalmau-Mena I, Joiner S, Linehan JM, O’Malley C,
Powell C, et al. Effect of fixation on brain and lymphoreticular vCJD
prions and bioassay of key positive specimens from a retrospective
vCJD prevalence study. J Pathol 2011; 223: 511–8.
Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch A,
et al. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet
1996; 347: 921–5.
Wroe SJ, Pal S, Siddique D, Hyare H, Macfarlane R, Joiner S, et al.
Clinical presentation and pre-mortem diagnosis of variant
Creutzfeldt-Jakob disease associated with blood transfusion: a case
report. Lancet 2006; 368: 2061–7.
Prion infectivity in subclinical vCJD Brain 2013: 136; 1139–1145 | 1145
    • "Importantly, prions replicate in tissues other than the nervous system, mainly lymphoid tissues. It is notably the case for vCJD and eventually the clinical phase could even not appear resulting in an asymptomatic carriage of vCJD infection [2,3]. To date, there is no certitude about asymptomatic vCJD prevalence. "
    [Show abstract] [Hide abstract] ABSTRACT: Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods - in vitro, ex vivo and in vivo assays - to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA) and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages). However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.
    Full-text · Article · Aug 2014
    • "For human prion diseases, the WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies (WHO, 1999) identified specific risk tissues for CJD. These guidelines were established before it was reported that peripheral organs such as the spleen and tonsils in vCJD patients may harbor significant levels of prion infectivity (Bruce et al., 2001; Peden and Ironside, 2004; Bishop et al., 2013), and that vCJD may have been transmitted by the transfusion of blood to blood-products from affected donors (Llewelyn et al., 2004; Wroe et al., 2006). The magnitude of prion infectivity may vary Risk of Pion Disease Transmission During Dissection 827 however, as a recent case of vCJD has been reported in which extremely low levels of prions were found in lymphoreticular tissues (Mead et al., 2014). "
    [Show abstract] [Hide abstract] ABSTRACT: Prion diseases (or transmissible spongiform encephalopathies) are a unique group of fatal progressive neurodegenerative diseases of the central nervous system. The infectious agent is hypothesized to consist solely of a highly protease-resistant misfolded isoform of the host prion protein. Prions display a remarkable degree of resistance to chemical and physical decontamination. Many common forms of decontamination or neutralization used in infection control are ineffective against prions, except chaotropic agents that specifically disrupt proteins. Human cadaveric prosection or dissection for the purposes of teaching and demonstration of human anatomy has a distinguished history and remains one of the fundamentals of medical education. Iatrogenic transmission of human prion diseases has been demonstrated from the inoculation or implantation of human tissues. Therefore, although the incidence of human prion diseases is rare, restrictions exist upon the use of tissues from patients reported with dementia, specifically the brain and other central nervous system material. A current concern is the potential for asymptomatic variant Creutzfeldt–Jakob disease transmission within the UK population. Therefore, despite the preventative measures, the transmission of prion disease through human tissues remains a potential risk to those working with these materials. In this review, we aim to summarize the current knowledge on human prion disease relevant to those working with human tissues in the context of anatomical dissection. Clin. Anat., 2014. © 2014 Wiley Periodicals, Inc.
    Full-text · Article · Apr 2014
  • Article · Apr 2013
Show more