Biochemical and Biophysical Research Communications xxx (xxxx) xxx

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Discrimination between L-type and C-type bovine spongiform

encephalopathy by the strain-specific reactions of real-time quaking-
induced conversion
Kaori Ubagai a, Shigeo Fukuda b, Tsuyoshi Mori c, Hanae Takatsuki c, Yuzuru Taguchi a,
Soichi Kageyama b, Noriyuki Nishida a, Ryuichiro Atarashi c, *
a
Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
b
The Committee of Animal Experiment, Animal Research Center, Hokkaido Research Organization, Shintoku, Hokkaido, Japan
c
Division of Microbiology, Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Real-time quaking-induced conversion (RT-QUIC) assays using Escherichia coli-derived purified recom-
Received 10 March 2020 binant prion protein (rPrP) enable us to amplify a trace amount of the abnormal form of PrP (PrPSc) from
Accepted 30 March 2020 specimens. This technique can be useful for the early diagnosis of both human and animal prion diseases
Available online xxx
and the assessment of prion contamination. In the present study, we demonstrated that there are strain-
specific differences in the RT-QUIC reactions between an atypical form of bovine spongiform encepha-
Keywords:
lopathy (BSE), L-BSE, and classical BSE (C-BSE). Whereas mouse rPrP (rMoPrP) was efficiently converted
Bovine spongiform encephalopathy
to amyloid fibrils in the presence of PrPSc seed derived from either L-BSE or C-BSE, hamster rPrP (rHaPrP)
Real-time quaking-induced conversion
Strain-specific differences
was converted only in L-BSE, not C-BSE. These characteristics were preserved in the second round re-
Recombinant prion protein action, but gradually weakened in the subsequent rounds and were completely lost by the fifth round,
most likely due to the selective growth advantage of nonspecific rPrP amyloid fibrils in the RT-QUIC. Our
findings further enhance the discrimination of prion strains using RT-QUIC, and further our under-
standing of the molecular basis of prion strains.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction resistant PrP (PrPres), respectively. C-BSE is caused by the con-

Prion diseases, also known as transmissible spongiform en-
cephalopathies (TSEs), are fatal neurodegenerative diseases char-
acterized by the accumulation of abnormal prion protein (PrPSc) in
the central nervous system and include CreutzfeldteJakob disease
(CJD) in humans, bovine spongiform encephalopathy (BSE) in cat-
tle, chronic wasting disease (CWD) in deer, and scrapie in sheep.
BSE can be categorized into two groups, namely, classical BSE (C-
BSE) and atypical forms of BSE, which include L-BSE and H-BSE
[1,2]. These two types of atypical BSE are classified based on the
apparent low and high molecular weights of proteinase K (PK)-
Abbreviations: RT-QUIC, real-time quaking-induced conversion; PrPSc, abnormal
form of prion protein; PrPres, proteinase K-resistant prion protein; BSE, bovine
spongiform encephalopathy; CJD, CreutzfeldteJakob disease; CWD, chronic wasting
disease; PK, proteinase K; ThT, thioflavin T; SD50, 50% seeding dose; rPrP, recom-
binant prion protein; rMoPrP, recombinant mouse prion protein; rHaPrP, recom-
binant hamster prion protein; BH, brain homogenate; CSF, cerebrospinal fluid.
* Corresponding author.
E-mail address: ryuichiro_atarashi@med.miyazaki-u.ac.jp (R. Atarashi).
sumption of contaminated meat and bone meal. C-BSE was once a
serious public health threat, but as a result of the successful
implementation of effective controls, its incidence has declined
significantly in recent years and is now estimated to be very low.
Atypical BSE is thought to occur spontaneously and sporadically,
with most cases confirmed in older cattle, primarily those 8 years
and older. Assuming that atypical BSE is sporadic that occurs
naturally at a low frequency, as with sporadic CJD, it is difficult to
eradicate atypical BSE.
Although cases of atypical BSE transmission have not been re-
ported in the natural environment, L-BSE can be experimentally
transmitted to non-human primates [3,4] and human PrP-
expressing transgenic mice [5e7]. Thus, L-BSE may be infectious
to humans. However, the transmission of H-BSE has not been
confirmed [7]. These results suggest that it is important for public
health to call attention to atypical BSE, especially L-BSE.
Real-time quaking-induced conversion (RT-QUIC) can efficiently
amplify and detect PrPSc in trace amounts in a test tube, and this
enables the early diagnosis of prion diseases including BSE [8e10].

https://doi.org/10.1016/j.bbrc.2020.03.183
0006-291X/© 2020 Elsevier Inc. All rights reserved.

Please cite this article as: K. Ubagai et al., Discrimination between L-type and C-type bovine spongiform encephalopathy by the strain-specific
reactions of real-time quaking-induced conversion, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/
j.bbrc.2020.03.183
2 K. Ubagai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Furthermore, it is suggested that C-BSE and atypical BSE can be
distinguished by RT-QUIC using various recombinant PrP (rPrP)
substrates without confirming the banding pattern of PrPSc by
western blotting [11e14]. These techniques are mainly based on the
time-dependent increase in fluorescence of thioflavin T (ThT) be-
tween various recombinant PrP substrates for the same prion strain
in the first round reaction. However, since the time taken for the
increase in fluorescence changes depending on the amount of PrPSc
and/or impurities in the sample, it may be difficult to differentiate
C-BSE from atypical BSE in practice.
In this study, we examined whether C-BSE and L-BSE can be
distinguished from each other by performing RT-QUIC continuously
from the second round onwards.
2. Materials and methods
2.1. Recombinant PrP purification
Recombinant PrP (rPrP) equivalent to residues 23e231 of the
mouse PrP and hamster PrP were expressed, refolded into a soluble
form, and purified essentially as previously described [8,15]. The
concentration of rPrP was determined by measuring the absor-
bance at 280 nm. The purity of the final protein preparations was

99%, as estimated by SDS-PAGE, immunoblotting. After purifica-
tion, aliquots of the proteins were stored at 80  C in 10 mM
phosphate buffer, pH6.8 or distilled water.
2.2. Preparation of brain homogenates and western blotting
C- and L-BSE infected cattle brain tissues [16,17]were homoge-
nized at 10% (w/v) in ice-cold PBS supplemented with a protease
inhibitor mixture (Roche, Basel, Switzerland) using a multi-bead
shocker (Yasui Kikai, Osaka, Japan). After centrifugation at
2000g for 2 min, supernatants were collected and frozen at
80  C until use. Total protein concentrations were determined by
the BCA protein assay (Thermo Fisher Scientific, MA, USA). Each
brain homogenate (BH) was analyzed by western blotting of 50 mg
equivalents of each sample. Briefly, each sample was digested with
20 mg/ml of PK at 37  C for 30 min, followed by boiling at 95  C for
5 min with sample buffer (2% SDS, 5% b-mercaptoethanol, 5% su-
crose, 0.005% bromophenol blue, 62.5 mM Tris-HCl, pH 6.8). Some
PK-digested samples were treated with PNGase F (New England
Biolabs, MA, USA) according to the manufacturer’s instructions.
Samples were separated by SDS-PAGE on SDS-polyacrylamide (15%)
gels and then transferred onto polyvinylidene difluoride (PVDF)
membranes (Merck Millipore, MA, USA) for 90 min at 300 mA. The
membranes were blocked with 5% skim milk in TBST (10 mM Tris-
HCl, pH 7.8, 100 mM NaCl, 0.1% Tween 20) for 1 h at room tem-
perature. The membranes were probed with monoclonal anti-PrP
antibody SAF84 (Cayman Chemical, MI, USA) at a 1:1000 dilution,
and then with horseradish peroxidase (HRP)-conjugated anti-
mouse IgG antibody at a 1:5000 dilution. Immunoreactive bands
were visualized using an ECL Western Blotting Detection Kit (GE
Healthcare, IL, USA).
All animal experiments were approved by the Committee of
Animal Experiment, Animal Research Center, Hokkaido Research
Organization, and were performed according to the Guideline for
Animal Experiment of the Ministry of Agriculture, Forestry, and
Fisheries of Japan.
2.3. Real-time quaking-induced conversion
Reaction mixtures were prepared in a 96-well optical black
bottom plate (Thermo Fisher Scientific) to a final total volume of
100 ml. The final concentrations of reaction buffer components were
Please cite this article as: K. Ubagai et al., Discrimination between L-type and C-type bovine spongiform encephalopathy by the strain-specific
reactions of real-time quaking-induced conversion, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/
j.bbrc.2020.03.183
500 mM NaCl, 25 mM PIPES pH 7.0, 1 mM EDTA, 10 mM Thioflavin T
and 50e80 mg/ml rPrP. The optimized concentration of rPrP was
determined for each lot. Five ml/well of diluted brain homogenate or
artificial cerebrospinal fluid (A-CSF, 125 mM NaCl, 2.5 mM KCl,
1 mM MgCl2, 2 mM CaCl2, 0.05% glucose and 0.2 ng/ml BSA) was
added as a seed or control the reaction solution. The 96-well plate
was covered with sealing tape (Thermo Fisher Scientific) and
incubated at 37  C in Infinite M200 plate reader (Tecan, Ma€nnedorf,
Switzerland) with intermittent shaking. The kinetics of amyloid
formation were monitored by reading the fluorescence intensity at
the bottom of the plate every 10 min with an excitation wavelength
of 440 nm and an emission wavelength of 485 nm. To compensate
for differences between fluorescent plate readers, the negative
control measurements were averaged, and the acquired data for
each sample were divided by the average of the negative control.
The 50% seeding dose (SD50) was calculated by the Spear-
maneKa €rber method [10,18].
3. Results
First, western blotting was performed to confirm that the C-BSE
and L-BSE samples used in this study showed strain-specific dif-
ferences in PrPres banding patterns after PK treatment (Fig. 1A). In
the brains of C-BSE-infected cattle, a band pattern of PrPres pre-
dominantly in the di-glycosylated form was observed, whereas in
the brains of L-BSE-infected cattle, the mono-glycosylated form was
predominant. Furthermore, PNGase F treatment showed that the
estimated molecular sizes were about 2-kDa smaller than those of
C-BSE (Fig. 1B).
Next, we tested the conversion efficiencies in RT-QUIC seeded
with either C-BSE or L-BSE using mouse rPrP (rMoPrP) or hamster
rPrP (rHaPrP) as a substrate. Although L-BSE showed no substantial
difference in the 50% seeding dose (SD50) of RT-QUIC between
rMoPrP and rHaPrP, C-BSE resulted in no conversion of rHaPrP into
the amyloid fibril form (Table 1 and Fig. 2A). To verify whether the
strain-specific non-reactivity of C-BSE to rHaPrP was maintained
after the second round, the second round of RT-QUIC was per-
formed using the positive reaction solution of the first round as a
Fig. 1. Western blotting of PrPres in midbrain tissues from either C-BSE or L-BSE. (A)
Brain homogenates were subjected to proteinase K digestion (20 mg/ml, 37  C, 30min)
followed by immunoblotting with SAF84 antibody. Lanes 1: uninfected cattle; Lanes 2
and 3: C-BSE (n ¼ 2); Lanes 4 and 5: L-BSE (n ¼ 2). (B) After consecutive treatments of
PK and PNGase F, samples were analyzed by western blotting. Molecular mass markers
are indicated in kilodaltons (kDa) on the left.
 K. Ubagai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 3

Table 1 show any fibril formation when rHaPrP was used as a substrate,
The 50% of seeding doses (SD50) in the brain homogenate derived from either C-BSE whereas all wells were ThT-positive in the reactions with rMoPrP.
or L-BSE-infected cattle.
By contrast, when rMo-fib [(1)/L-BSE] or rHa-fib [(1)/L-BSE] derived
C-BSE L-BSE from L-BSE were used as a seed, fibril formation occurred efficiently
rMoPrP 6.94 ± 0.66 8.15 ± 0.14 with both rMoPrP and rHaPrP (Fig. 2B). In the third round, rHaPrP
rHaPrP 0 7.30 ± 1.11 again resisted fibril formation against rMo-fib [(2)/C-BSE]. In the
The SD50 (log SD50/g brain) values are the means ± S.D. from four independent fourth round, one out of four wells became ThT-positive following
experiments. reactions with rHaPrP. In the fifth round, all wells became positive
following reactions with rHaPrP. Additionally, when the solution
consisting of rHaPrP from a well that was positive in the fourth
seed (Fig. 2B). For comparison with C-BSE, the positive reaction round (rHa-fib [(4)/C-BSE]) was used as a seed in the fifth round,
solution of rMoPrP or rHaPrP substrates seeded with L-BSE in the both rMoPrP and rHaPrP revealed fibril formation with high effi-
first round was also used. In the second round, rMoPrP fibrils ciency (Fig. 2C).
seeded with C-BSE in the first round (rMo-fib [(1)/C-BSE]) did not

Fig. 2. Sequential RT-QUIC analysis initially seeded with C-BSE or L-BSE. (A) The first round of RT-QUIC using rMoPrP or rHaPrP was examined. The type seed used is shown on the
left side of the graphs. The colored curves represent the kinetics of ThT fluorescence from an individual reaction seeded with C-BSE (2  105 dilution) or L-BSE (1  106 dilution).
(B) The second round of RT-QUIC seeded with the 0.5% (v/v) solution from a positive well of the first round was examined. (C) The third, fourth, and fifth round of RT-QUIC were also
examined. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: K. Ubagai et al., Discrimination between L-type and C-type bovine spongiform encephalopathy by the strain-specific
reactions of real-time quaking-induced conversion, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/
j.bbrc.2020.03.183
4 K. Ubagai et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
4. Discussion
As shown in previous and current studies, PrP sequence
dependence (species barrier) in RT-QUIC reactions is low compared
with animal and cell culture experiments. This is probably because
rPrP has no glycosylation modification or GPI-anchor modification,
and/or that there is increase in the unfolding of the rPrP confor-
mation resulting from stirring of the reaction solution. In animal
transmission experiments, C-BSE was transmitted to mice but not
to Syrian hamsters [19], whereas, conversely, L-BSE was transmitted
to Syrian hamsters but not to mice [20,21]. By contrast, in the
present RT-QUIC studies, L-BSE efficiently induced both rMoPrP and
rHaPrP fibril formation, whereas C-BSE showed poor induction of
rHaPrP fibril formation. In other words, for C-BSE, the results of RT-
QUIC were consistent with the animal experiments, but for L-BSE,
the results differed. This suggests that the conversion of hamster
PrP to the C-BSE type-PrPSc structure was more strictly dependent
on the PrP sequence. The hamster PrP sequence differs from the
mouse PrP sequence by 9 amino acids, with the exception of the
signal sequence. It remains to be determined which amino acid
sites in hamster PrP are responsible for the resistance to fibril for-
mation seeded with C-BSE.
We reported previously that the strain specificities observed in
the first round of RT-QUIC, such as FTIR spectroscopic character-
ization and conformational stability, were lost in subsequent
rounds [15]. In the previous study, the strain-specific properties
were almost completely lost in the second round, whereas in this
study, the resistance to fibril formation of rHaPrP seeded with C-
BSE was maintained in the second round. Except for these subtle
differences, the two studies are essentially consistent in that
nonspecific fibrils, which have no strain-specific properties,
become gradually dominant in the RT-QUIC environment. This may
be explained by the paucity of hypothetical cofactors to maintain
strain-specific properties and/or a selective growth advantage of
nonspecific fibrils [22].
Based on these results, we propose the following C-BSE and L-
BSE discrimination methods. As shown in Fig. 3, in the first round,
RT-QUIC reactions are performed using only rMoPrP as a substrate
to determine the presence or absence of BSE prion infection in the
specimens derived from cattle. Next, in the second round (confir-
mation round), both rMoPrP and rHaPrP are used as substrates to
determine whether the prion activity comes from C-BSE or L-BSE.
Fig. 3. A schema of the differential diagnosis between C-BSE and L-BSE using RT-QUIC.
In the first round, only rMoPrP is used to verify the presence of prion activity in
suspected BSE samples. If positive, a second round using rMoPrP or rHaPrP is per-
formed to determine whether it is atypical BSE (L-BSE) or C-BSE.
Please cite this article as: K. Ubagai et al., Discrimination between L-type and C-type bovine spongiform encephalopathy by the strain-specific
reactions of real-time quaking-induced conversion, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/
j.bbrc.2020.03.183
While the sample used in the first round, such as cerebrospinal
fluid, blood, urine or tissue homogenate, generally contains impu-
rities that affect the RT-QUIC reaction, such contaminants are
avoided in the second round. This would be the advantage of
determining the prion strain in the second round.
Unfortunately, H-BSE was not available in this study and could
not be examined. However, according to the study of Masujin et al.,
rHaPrP was ThT-positive when seeded with H-BSE [12]. Therefore,
it is highly likely that C-BSE and atypical BSE can be distinguished
by the method presented in this study. The development of a
method for distinguishing H-BSE and L-BSE in the second round
using a recombinant PrP other than rHaPrP is a future research
topic.
In conclusion, we have shown that there are strain-specific
differences in the reactivity to rHaPrP even in the second round
of RT-QUIC between C-BSE and L-BSE. Our findings provide another
means with which to discriminate between C-BSE and L-BSE using
an RT-QUIC assay.
Author contributions
K$U., N.N. and R.A. designed the entire project. K.U. and R.A.
wrote the manuscript. K.U., T.M., H.T., Y.T. and R.A. performed the
experiments and analyzed the data. S.F. and S.K. contributed to the
collection of bovine specimens and provided information about
subjects. R.A. and N.N. supervised and discussed the data.
Funding
This work was supported by a Health Labour Sciences Research
Grant (Study on risk management of livestock and poultry diseases
through food: 201823009A); a grant form Takeda Science Foun-
dation, Japan.
Declaration of competing interest
There are no potential conflicts of interest to disclose.
Acknowledgments
The authors thank Dr. Takayuki Fuse for helpful discussions, and
Atsuko Matsuo for technical assistance. We also thank Kate Fox,
DPhil, Edanz Group (www.edanzediting.com/ac) for editing a draft
of this manuscript.
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