Journal of Biosafety and Biosecurity 2 (2020) 31–35

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Journal of Biosafety and Biosecurity

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

Inactivation of severe fever with thrombocytopenia syndrome virus for

improved laboratory safety
Toshihiko Harada a, Shuetsu Fukushi b,⇑, Takeshi Kurosu b, Tomoki Yoshikawa b, Masayuki Shimojima b,
Kiyoshi Tanabayashi a, Masayuki Saijo b
a
Division of Biosafety Control and Research, National Institute of Infectious Diseases, Tokyo, Japan
b
Department of Virology 1, National Institute of Infectious Diseases, Tokyo, Japan

a r t i c l e i n f o s u m m a r y

Article history: Severe fever with thrombocytopenia syndrome (SFTS), a tick-borne infectious disease with high mortal-
Received 9 September 2019 ity, is diagnosed by the serological testing of serum samples from patients in the acute and convalescent
Received in revised form 10 January 2020 phases and/or by direct detection of the SFTS virus (SFTSV). Conventionally, heat and UV treatments have
Accepted 9 February 2020
been used to inactivate the viruses present in serum samples before performing the serological assays.
Here, we examined the inactivation conditions optimal for SFTSV-containing serum samples to ensure
the safety of laboratory workers while maintaining the accuracy of serological assay results simultane-
Keywords:
ously. Heating human serum samples spiked with SFTSV to 60 °C for 30 min or exposing them to UV irra-
Biosafety
Inactivation
diation for 30 min, reduced the infectious virus titer below the limit of detection. SFTSV in sera from
Severe fever with thrombocytopenia patients in the acute phase of SFTS was completely inactivated by heating to 60 °C for 30 min, followed
syndrome (SFTSV) by UV irradiation for 10 min. This inactivation procedure had minimal impact on the performance of
Serology SFTSV antibody detection tests. The data provided herein can serve as a guide for laboratory workers
and researchers working with SFTS serological tests.
Ó 2020 Published by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction accuracy of neutralization assays.2 A previous study investigating

Body secretions and excretions, blood, serum, and tissue sam-
ples from patients infected with highly pathogenic viruses may
contain infectious virus particles that pose serious risks to health-
care and laboratory workers.1 Thus, clinical samples should be
treated appropriately to inactivate pathogens and prevent
laboratory-acquired infections. In addition, the procedures used
to inactivate potential pathogens should have a minimal effect
on the accuracy of subsequent diagnostic assays.
Typically, serum samples from patients with viral infections are
tested in antibody-based diagnostic assays such as IgG/IgM
enzyme-linked immunosorbent assays (ELISAs), immunofluores-
cence antibody assays (IFAs), and neutralization tests. Serum sam-
ples are typically treated first by heat inactivation at 56 °C for
30 min to minimize the effects of complement on the results of
antibody-based assays. Additionally, heat inactivation of serum
samples by heating to 56 °C for 30 min is the recommended
method for eliminating infectious dengue virus to ensure the
⇑ Corresponding author at: Department of Virology 1, National Institute of
Infectious Diseases, 1-23-1, Shinjyuku, Tokyo 162-8640, Japan.
E-mail address: fukushi@nih.go.jp (S. Fukushi).
the thermal stability of cell culture-derived hepatitis C virus
indicated that heating to 56 °C for 40 min is required for the elim-
ination of infectious virus.3 However, other studies have shown
that a longer incubation time (e.g., 60 min) at 56 °C or incubation
at a higher temperature is required for the complete inactivation of
many viruses.4–8 Due to the different thermal stabilities of various
pathogenic viruses, evidence-based inactivation procedures are
required to reduce the risk to laboratory workers.
In addition to heat inactivation, biological samples can be trea-
ted with ultraviolet (UV) light or chemical disinfectants.9–11
Although UV irradiation is effective in causing damage to the viral
genome, the optimum exposure time required to eliminate viral
infectivity varies according to the solvent component of the virus
preparation (e.g., culture medium or serum).3 Severe fever with
thrombocytopenia syndrome (SFTS), which is caused by a novel
Phenuivirus called Huaiyangshan banyangvirus (herein referred to
as SFTS virus (SFTSV)), is a tick-borne infectious disease with a high
mortality rate in China, South Korea, and Japan.12 A retrospective
study indicated that SFTS is endemic to Vietnam.13 The clinical
symptoms of SFTS include fever, nausea, and vomiting. Total blood
cell counts revealed thrombocytopenia and leukopenia. As the dis-
ease progresses, patients develop hemorrhagic and neurological

https://doi.org/10.1016/j.jobb.2020.02.002
2588-9338/Ó 2020 Published by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
32 T. Harada et al. / Journal of Biosafety and Biosecurity 2 (2020) 31–35
symptoms, disseminated intravascular coagulation, and multiple
organ failure.12 Similar to other viral hemorrhagic fevers,14,15 the
viral load in the serum of SFTS patients often exceeds 1.0  106
(e.g., up to 1.0  1010) copies per mL.16
The laboratory diagnosis of SFTS is undertaken by performing
molecular and antigen detection tests, as well as antibody detec-
tion assays using blood samples from patients in the acute and
convalescent phases of disease.16,17 Antibodies specific for SFTSV
can be detected in serum samples using IFA, ELISA, or neutraliza-
tion tests. In standard laboratory settings, these serological assays
must be performed after inactivating blood samples, since SFTSV
infection may result through exposure to viruses in these samples.
Heat and UV treatment are commonly used to inactivate viruses.
However, the optimal conditions required to ensure the inactiva-
tion of SFTSV and the safety of laboratory workers are unclear.
The need to inactivate the virus must be balanced with the main-
tenance of antibody titers in treated samples—viral inactivation
must not affect antibody titers to ensure that the accuracy of the
serological test results is maintained.
Therefore, the objective of this study was to determine the opti-
mal heat and UV treatment conditions required to inactivate SFTSV
in serum samples and assess their effects on the performance of
antibody diagnostic tests for SFTS.
2. Materials and methods
2.1. Cells and viruses
Monkey kidney-derived Vero cells were obtained from Ameri-
can Type Culture Collection (Summit Pharmaceuticals Interna-
tional, Japan) and cultured at 37 °C in Dulbecco’s modified
Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO) supple-
mented with 5% heat-inactivated fetal bovine serum (FBS, Thermo-
Fisher Scientific, Rockford, IL) and antibiotics (10 units/mL
penicillin and 10 mg/mL streptomycin, ThermoFisher Scientific).
SFTSV YGI strain was propagated in cultured Vero cells.17 Experi-
ments using infectious SFTSV were conducted in a biosafety level
(BSL)-3 laboratory at the National Institute of Infectious Diseases.
2.2. Clinical samples
Serum samples A and B were obtained from two SFTS patients
in the acute phase, and serum samples C and D were obtained from
two SFTS patients in the convalescent phase. These serum samples
were collected for the diagnosis of SFTS in Japan. The viral genome
copy numbers in samples A and B, as determined by quantitative
RT-PCR,16 were 1.0  106.4 and 1.0  107.7 per mL, respectively.
The anti-SFTSV-IgG antibody titer in samples C and D, as deter-
mined in an immunofluorescence antibody assay,17 was more than
640. All protocols and procedures were approved by the research
ethics committee of the National Institute of Infectious Diseases
for the use of human subjects (no. 982).
2.3. UV irradiation and heat treatment of SFTSV-spiked samples
SFTSV strain YG1 (3.2  107 TCID50/mL) was diluted 10-fold in
DMEM supplemented with 2% FBS (2% FBS-DMEM) or pooled
human serum (Tennessee Blood Services, Memphis, TN) to prepare
virus samples (3.2  106 TCID50/mL) for the inactivation experi-
ments. Next, 200 mL of virus sample was aliquoted into a 1.5 mL
transparent polypropylene tube (No.72.692S, Sarstedt, Nümbrecht,
Germany). The virus samples were then heat-treated at 56 °C or
60 °C for 5–60 min or UV-irradiated (wavelength, 312 nm; output
power, 2.5 mW/cm2) for 5 s to 30 min using a transilluminator
(model TPP-10 M, Vilber-Lourmat, France). The distance between
the UV source and samples was approximately 1.0 cm and the
UV intensity was 860 mW/cm2. All samples were stored at 80 °C
until virus titration.
2.4. Virus titration
The titration of SFTSV in heat- or UV-treated samples was per-
formed as previously described.18 Briefly, Vero cells seeded onto a
96-well microplate were inoculated with virus samples (serially
diluted 10-fold) and incubated at 37 °C/5% CO2 for 4–5 days. The
cells were then fixed with 10% formalin. To detect cells containing
replicating SFTSV, viral antigens were stained with a rabbit anti-
SFTSV nucleocapsid (N) antibody,19 followed by Alexa Fluor 488
conjugated-goat anti-rabbit IgG (ThermoFisher Scientific). The
viral titer was determined as the 50% tissue culture infectious dose
(TCID50).
2.5. Inactivation of clinical serum samples
Serum samples A and B (obtained from SFTS patients in the
acute phase) were heat-treated and/or UV-irradiated as follows:
50 mL of serum in 1.5 mL transparent polypropylene tubes were
incubated at 56 °C for 30 min, 60 °C for 30 min, or 60 °C for
60 min, followed by exposure (or not) to UV irradiation for
10 min. Serum samples were then inoculated with Vero cells
seeded onto a 12-well plate. The cells were incubated in 2% FBS-
DMEM at 37 °C/5% CO2 for 24 h. Next, the supernatant was
replaced with fresh 2% FBS-DMEM and the cells were incubated
for an additional 6 days. The cells were passaged into new flasks
every 7 days (approximately). This blind passage was repeated
thrice (inoculated cells were cultured for 28 days in total). To test
whether the virus was inactivated, a small aliquot of passaged cells
was seeded onto a 24-well plate and incubated for 3 days before
fixing and staining as described above (Subsection 2.4). Serum
samples C and D (obtained from SFTS patients in the convalescent
phase) were treated with heat and/or UV radiation as follows:
50 mL of serum in 1.5 mL transparent polypropylene tubes was
incubated at 60 °C for 30 min or at 60 °C for 60 min, followed by
exposure to UV irradiation (or not) for 10 min. SFTSV-IgG titers
in serum samples were determined in an IgG ELISA as previously
described.20
3. Results
3.1. Inactivation of SFTSV
To examine how the infectivity of SFTSV was affected by heat
treatment, pooled human serum was spiked with SFTSV prior to
heating at 56 °C or 60 °C for 5–60 min. The viral titers were then
determined (Fig. 1A). The viral titers remained high at more than
104 TCID50/mL and 103 TCID50/mL after heating to 56 °C for
30 min and 60 min, respectively. In contrast, heating to 60 °C for
30 min led to a marked reduction in viral titer; no infectious SFTSV
particles were detected (Fig. 1A). Next, we examined the degree of
SFTSV inactivation after UV irradiation (Fig. 1B). SFTSV (spiked into
pooled human sera) was exposed to UV irradiation for 5 s to
30 min. Exposure times of less than 1 min had no effect on SFTSV
infectivity, whereas an approximately 2-log reduction in the SFTSV
titer was observed after 10 min. No infectious SFTSV particles were
detected after 30 min UV irradiation. Since serum proteins (e.g.
serum albumin, transferrin, and immunoglobulin) may interfere
with the effect of UV light on viruses, we mixed the SFTSV stock
solution with 2% FBS-DMEM to examine the tolerance of SFTSV
to UV irradiation. Under these conditions, the SFTSV titer
decreased below the limit of detection after 1 min, indicating that
 T. Harada et al. / Journal of Biosafety and Biosecurity 2 (2020) 31–35 33

Table 1
Heat inactivation of SFTSV in serum from SFTS patients.

17 dpi 24 dpi 35 dpi

Sample A
Uninfected
4 °C, 60 min + + +
56 °C, 30 min +/+ N.T./N.T. N.T./N.T.
60 °C, 60 min / / /
Sample B
Uninfected
4 °C, 60 min + + +
56 °C, 30 min +/+ N.T./N.T. N.T./N.T.
60 °C, 60 min +/ +/ N.T./

+SFTSV antigen-positive.
SFTSV antigen-negative.
N.T., not tested; dpi, days post-infection.

in cells at 17 dpi). A single heat treatment at 60 °C for 60 min was

thus insufficient to inactivate SFTSV completely. Therefore, we
tested the efficacy of combined heat and UV treatments. First, we
examined the efficacy of UV irradiation for 10 min after heat treat-
ment of serum samples, since this protocol reduced the virus titer
in spiked samples by approximately 2-log (see Fig. 1B). No virus
antigen was detected in the cells inoculated with serum heated
to 60 °C for 30 min followed by UV irradiation for 10 min, or after
heating to 60 °C for 60 min followed by UV irradiation for 10 min
(Table 2). This indicates that heating to 60 °C for 30 min followed
by UV irradiation for 10 min inactivates SFTSV in patient serum
completely.

3.3. Effect of inactivation treatment on the sensitivity of ELISA

Fig. 1. Heat- or ultraviolet irradiation-inactivation of SFTSV. (A) Heat inactivation of
SFTSV in 90% human serum. Aliquots (200 mL) of SFTSV (3.2  106 TCID50/mL) were
treated at 56 °C (gray) or 60 °C (black) for the indicated time periods. Treated
samples were inoculated onto Vero cells for virus titration. (B) Inactivation of SFTSV
in 2% FBS-DMEM (gray) or 90% human serum (black) by UV irradiation. Aliquots
(200 mL) of SFTSV (3.2  106 TCID50/mL) in 90% human serum or 2% FBS-DMEM
were exposed to UV irradiation for the indicated time periods. Virus titers (TCID50/
mL) in the treated samples are shown. All assays were performed in quadruplicates.
The limit of detection (cut-off value, 101.75 TCID50/mL) is shown as a dashed line. N.
T., not tested.
UV treatment reduced SFTSV infectivity in the absence of excess
serum proteins (Fig. 1B). Based on these results, we concluded that
heating to 60 °C for 30 min or UV irradiation for 30 min was
required to inactivate SFTSV in spiked serum samples.
Sera from two convalescent SFTS patients (samples C and D)
were heat-treated with or without UV irradiation to examine
whether the inactivation treatment had a negative impact on anti-
body detection using an IgG ELISA. The absorbance values for sera
treated by heating to 60 °C for 30 min followed by UV irradiation
for 10 min were more than 90% and 80% (at a dilution of
100- and 400-fold, respectively) of that of untreated samples.
Heating serum sample C at 60 °C for 60 min followed by UV irradi-
ation for 10 min led to absorbance levels decreasing by 90% and
80% (after 100- and 400-fold dilutions, respectively) of those
obtained for untreated serum (Fig. 2A). These results suggest that
heating serum samples at 60 °C for 30 min followed by UV irradi-
ation for 10 min is the most effective method for inactivating
SFTSV with minimal impact on the sensitivity of the ELISA used
to detect anti-SFTSV antibodies.

3.2. Inactivation of patient serum

To test whether the above conditions were effective against Table 2

Heat- and UV-inactivation of SFTSV in patient serum.
SFTSV in clinical samples collected from SFTS patients, we treated
samples A and B (1  106.4 and 1  107.7 SFTSV genome copies/mL, 10 dpi 17 dpi 24 dpi 35 dpi
respectively) by heating to 56 °C for 30 min or to 60 °C for 60 min. Sample A
Control samples were not treated. Next, Vero cells were inoculated Uninfected
with these treated (or untreated) sera and passaged thrice. SFTSV 4 °C, 60 min + N.T. N.T. N.T.
60 °C, 30 min + UV 10 min / / / /
antigen was detected by IFA using an anti-SFTSV N antibody
60 °C, 60 min + UV 10 min / / / /
(Table 1). At 17 days post-inoculation (dpi), virus antigen was
Sample B
detected in cells inoculated with the samples treated at 56 °C for
Uninfected
30 min, indicating that the standard procedure used to inactivate 4 °C, 60 min + N.T. N.T. N.T.
complement (heating to 56 °C for 30 min) was insufficient to inac- 60 °C, 30 min + UV 10 min / / / /
tivate SFTSV. No virus antigen was detected in sample A after heat- 60 °C, 60 min + UV 10 min / / / /
ing at 60 °C for 60 min (no antigen was detected up to 35 dpi). + SFTSV antigen-positive.
However, one duplicate from sample B contained infectious virus SFTSV antigen-negative.
even after heating at 60 °C for 60 min (virus antigen was detected N.T., not tested; dpi, days post-infection.
34 T. Harada et al. / Journal of Biosafety and Biosecurity 2 (2020) 31–35
Fig. 2. Effect of virus inactivation procedures on the results of serological tests.
Serum samples C and D (shown in (A) and (B), respectively) were treated as
indicated. Anti-SFTSV antibody titers in diluted sera (1:100, gray bar; 1:400, black
bar) were measured in an IgG ELISA. The vertical axis indicates the relative optical
density (O.D.) of each sample (the O.D. of the untreated sample was set as 100%).
4. Discussion
Here, we deduced the optimal conditions required to inactivate
SFTSV to ensure the safety of laboratory workers without affecting
the accuracy of serological assays. First, we used human serum
spiked with SFTSV. We found that heating to 60 °C for 30 min
reduced the virus titer to undetectable levels. These observations
are in agreement with previously published data,4 which used
virus preparations with titers of 1  105–1  107 PFU/mL to
demonstrate that the time required to inactivate highly pathogenic
viruses (Lassa, Ebola, and Marburg virus) spiked into serum was
22–37 min at 60 °C. The previous study recommended that the
appropriate time, with an added margin of safety, was 60 min.
Under these conditions, biochemical laboratory tests that measure
serum sodium, potassium, chloride, glucose, and urea nitrogen
levels were unaffected.4 Furthermore, the WHO suggests that
treating specimens at 60 °C for 60 min is sufficient to inactivate
potentially infectious samples containing the Ebola virus.21
UV irradiation is another method commonly used to inactivate
viruses. We found that the time required to inactivate SFTSV in
culture medium was shorter than that required to inactivate the
virus in human serum (Fig. 1B). These data clearly indicate that
serum proteins affect the absorption of UV light by virus particles.
Given that factors other than serum proteins (e.g., the material of
the container/tube and the specification of the UV light source)
may also affect UV light absorption, optimum UV irradiation times
may vary according to the characteristics of laboratory instruments
or equipment. Under the experimental conditions described
herein, which used transparent polypropylene tubes and a speci-
fied UV source, we noted a marked reduction in SFTSV titer (unde-
tectable levels) after exposure to UV radiation for 30 min.
An important finding of our study is that heat treatment alone is
not sufficient to ensure the complete inactivation of SFTSV in
patient samples (Table 1). However, the experiments based on
SFTSV-spiked human serum showed that heating to 60 °C for
30 min was enough to reduce the viral titer to undetectable levels.
We found that for patient samples, additional treatment with UV
irradiation was required to completely inactivate residual infec-
tious SFTSV particles (Table 2). Since heating at 60 °C for 30 min
had minimal impact on the sensitivity of subsequent ELISAs, and
heating to 60 °C for 60 min reduced the sensitivity of the ELISA
(Fig. 2), we recommend the combined treatment of patient serum
by heating at 60 °C for 30 min followed by UV irradiation for
10 min.
Using heat to inactivate viruses has been speculated to damage
both glycoproteins and the viral genome, whereas UV irradiation
damages only the viral genome.11 In addition, different viruses
(e.g., enveloped/non-enveloped viruses or viruses with
segmented/non-segmented genomes) may show different thermal
stabilities.8 Therefore, evidence-based inactivation procedures are
required to reduce risks to laboratory staff. The data provided here
may serve as guidance for laboratory workers and researchers
working on serological diagnosis, particularly that of infectious dis-
eases caused by SFTSV.
CRediT authorship contribution statement
Toshihiko Harada: Investigation, Data curation, Writing - orig-
inal draft. Shuetsu Fukushi: Validation, Resources, Funding acqui-
sition, Writing - review & editing. Takeshi Kurosu: Resources,
Visualization. Tomoki Yoshikawa: Resources, Visualization.
Masayuki Shimojima: Methodology, Resources, Visualization.
Kiyoshi Tanabayashi: Supervision. Masayuki Saijo: Conceptual-
ization, Funding acquisition, Writing - review & editing.
Acknowledgment
This work was supported in part by a grant from the Japan
Agency for Medical Research and Development (AMED) (#
JP19fk0108070, JP19fk0108081, and JP19fk0108072).
Conflicts of interest
None declared.
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