Open Journal of Veterinary Medicine, 2012, 2, 120-123 Published Online September 2012 (
Binary Ethylenimine Inactivated Japanese Encephalitis
Virus Antigen Reveals Hemagglutination
Dong-Kun Yang*, Ha-Hyun Kim, Jin-Ju Nah, Kyung-Woo Lee, Jae-Young Song
Animal, Plant and Fishery Quarantine Inspection Agency (MIFAFF), Anyang, Korea
Email: *
Received June 28, 2012; revised July 23, 2012; accepted July 31, 2012
Severe climate change and global warming may impact significantly on vector-borne disease including Japanese en-
cephalitis (JE) infection in human and animals. Thus, veterinary authority requires large quantity of diagnostic tools to
survey vector-borne diseases. New producing method having a relation with JE antigen is needed to substitute conven-
tional sucrose-acetone extraction method using suckling mouse. So, we developed new manufacturing method using
polyethylene glycol (PEG) precipitation. Japanese encephalitis virus (JEV) was propagated in roller bottle containing
Vero cell and inactivated with two kinds of inactivating reagents. Viability of the supernatant of bulk containing antigen
was checked using Vero cell after inactivation. The supernatant did not show hemagglutination (HA) activity with
goose erythrocytes. The antigen inactivated by binary ethylenimine (BEI) and concentrated by PEG precipitation me-
thod was found to be 2048 HA, but the antigen inactivated by 0.3% formaldehyde solution and concentrated by PEG
precipitation method did not show HA titer. The antigen prepared from mice brain using sucrose-acetone extraction
method showed 256 HA titer. This BEI inactivation method does not evoke animal welfare problem and can replace the
conventional method that required biological hazardous reagents and suckling mice in preparing HA antigen. This new
BEI inactivation method was safe in producing HA antigen against JEV in laboratory and can reduce environmental
contamination of acetone.
Keywords: Hemagglutination; JEV; BEI Inactivation
1. Introduction
Climate change through recent global warming and ex-
treme weather may have significantly impacted the
transmission of vector-borne diseases such as Japanese
encephalitis virus (JEV), West Nile virus (WNV), and
tick-borne encephalitis virus (TBEV), as rapid weather
and climate changes can directly or indirectly affect mi-
gratory birds and mosquitoes. Transmission of JEV is
well kept in a mainly rural zoonotic cycle between mos-
quitoes and wild birds including egrets and herons [1,2].
Approximately 67,900 Japanese encephalitis (JE) cases
occur annually in Asian countries and about 50% of JE
cases occur in China [3]. JE outbreak occurs in children
aged 0 - 14 years. Most infections in human are inappar-
ent or produce a mild systemic illness with fever, head-
ache or asceptic meningitis. Inflammation of the brain,
spinal cord and meninges can cause stupor, tremors,
convulsions (especially in infants), spastic paralysis,
coma and death [4]. Patients infected with JE show hear-
ing and speech impaired judgment in recovery period.
JEV can also lead to infection in several animals such as
reptiles, pigs, horses, chickens, breeding birds such as her-
ons. In particular, livestock pigs infected with JEV can
amplify the JEV in body and are known as an amplifying
host [5]. Fattening pigs bitten by mosquitoes holding
JEV show viremia for a short period of time with sub-
clinical signs. However, pregnant sows with JEV infec-
tion cause still birth, mummified fetus, weaken piglets
showing neurological symptoms. Boars are also infected
with JEV that penetrates boar’s genital organ and make
the boar reduce a number of sperm and deform sperms
resulting in dropping pregnancy rate. Horse infected with
JEV naturally shows lack of appetite, fever, neurological
symptoms and pass away in the long run [6]. Veterinary
authorities in South Korea have been tried to immunize
livestock to block JE epidemic as well as to block the
influx and to predict the disease in pigs. Since 1990, pigs
being raised on farm have been vaccinated against JE.
And since 2001, sero-surverveillance for JEV has been
carried out over 9000 pigs every year.
When the blood-agglutinating viruses such as porcine
parvovirus (PPV), encephalomyocarditis virus (EMCV)
are propagated in cell culture systems and used as a he-
magglutinating antigen, they still possess hemagglutinat-
*Corresponding author.
opyright © 2012 SciRes. OJVM
D.-K. YANG ET AL. 121
ing activity. However, when JEV is cultivated and pro-
pagated in cell culture system, the supernatant of the JEV
does not have hemagglutinating activity. Therefore, he-
magglutinating antigens prepared from suckling mice
brains infected with JEV using the sucrose-acetone ex-
tracting method has been used in checking immune status
of JEV in pigs. However, the sucrose-acetone extracting
method is biologically hazardous in the process to ma-
nufacturers and has caused a debate about animal welfare.
Thus, new method to produce JEV antigen without using
suckling mice has been demanded in laboratory. In this
study, we report a new method to make highly concen-
trated JEV HA antigen using cell culture system.
2. Material and Methods
2.1. Propagation of Virus and Cells
The strain of JEV used as an antigen for the hemmagglu-
tination inhibition (HI) test was KV1899. This strain iso-
lated from Korean pig blood in 1999 was propagated
using Vero cells cultured in α-minimum essential me-
dium (MEM; Gibco BRL, USA) supplemented with an-
tibiotics (100 IU/mL penicillin and 100 µg/mL strepto-
mycin), an antimycotic (0.25 µg/mL amphotericin B),
and 5% fetal bovine serum (FBS; Gibco BRL). After
inoculation, when 90% of cytopathic (CPE) effects in
cells were shown, the roller bottles were frozen at 70˚C
and thawed three times.
2.2. Inactivation of Antigen
The JEV was inactivated with binary ethylenimine (BEI)
by method of Larghi et al. [7]. In brief, BEI was prepared
from 2% 2-bromo-ethylamine hydrobromide (2-BEA,
Sigma, USA) in solution of 0.2 N NaOH, and the solu-
tion was treated in water bath at 37˚C for 1 hr, and then
was adjusted to 0.1 M BEI. The final concentration of
BEI was adjusted to 0.001 M of bulk and pH of bulks
also was adjusted to 8.0 with 1 N NaOH. Inactivation
was done at 37˚C for 10 hrs. As another inactivation
method, 37% of formaldehyde solution (Junsei chemicals
Ltd., Japan) was added to viral suspensions to make final
formaldehyde concentration equal to 0.3% [8]. Inactiva-
tion of the JEV by formaldehyde was also done at 37˚C
for 10 hrs. Vero cells were employed to check whether
the viruses were inactivated or not. After checking the
inactivation of viruses, bulks were used for preparation
of high concentrated HA antigen.
2.3. Polyethylene Glycol Precipitation
The inactivated JEV was centrifuged at 3000 × g to re-
move cell debris and concentration by polyethylene gly-
col (PEG) precipitation was carried out essentially as
described by Lewis and Metcalf (1988), with some modi-
fication [9]. Sodium chloride was added to obtain a final
concentration of 0.5 M and PEG (Sigma, USA, MW
8000) was added to a final concentration of 8%. The an-
tigen was placed at 4˚C for 10 hrs for the precipitation of
virus and then centrifuged at 10,000 × g for 30 minutes.
The supernatant medium was poured off leaving the cen-
trifuged tubes inverted until the medium has been re-
moved. The precipitate was suspended in 4% borate buf-
fered saline (BBS: pH 9.0) or phosphate buffered saline
(PBS: pH 7.2) using 1% of the original volume. The high
concentrated antigen was stored at 70˚C.
2.4. Preparation of Viral Antigen with
Mice Brain
The mice inoculation experiment described in this study
had been approved by the Animal Ethics Committee of
our institute (Animal Ethics number : QIA-AEC-2010-44)
antigen using a sucrose—acetone extraction method was
also prepared from the brains of suckling mice infected
with the Korean isolate of JEV strain KV1899 [10,11].
Briefly, JEV was inoculated into the brain of mice aging
3 to 4 days. When the inoculated mice began to show
signs of paralysis, the mice were euthanized by CO2 and
frozen at 70˚C. The infected suckling mice-mouse brain
was homogenized with 5 volumes of 8.5% sucrose solu-
tion and the homogenate was added to 20 volumes of
chilled acetone. After shaking vigorously, the milky su-
pernatant was discarded and an equal volume of acetone
was added to the flask. This preparation was incubated
for 1 hr at 4˚C to dehydrate the sediment. The super-
natant was discarded and the sediment was dried using a
vacuum pump. The dried antigen was suspended in saline.
After centrifugation for 10 min at 8000 × g, the super-
natant was used as antigen for hemagglutination assay.
2.5. Hemagglutination Assay (HA) Test
An HA test to check HA titer of concentrated JEV anti-
gen was performed in 96-well microplates, using slightly
modified standard methods [10]. In brief, 50 μL of con-
centrated antigen was diluted with 0.4% borate saline
solution. After dilution, 50 μL of 0.33% goose erythro-
cytes were added, and the microplates were incubated at
room temperature for 30 minutes. The HA titer was ex-
pressed as the reciprocal of the highest dilution of anti-
gen showing complete hemagglutination.
3. Results
The JEV propagated in roller bottle containing Vero cell
was inactivated with two kinds of inactivates. After inac-
tivation, some part of bulk was dialyzed in phosphate
buffer. Both supernatants inactivated with both 0.3%
formaldehyde and 0.001 M BEI did not exhibit specific
Copyright © 2012 SciRes. OJVM
CPE in Vero cells, respectively. The supernatants did not
reveal HA activity with goose erythrocytes (Table 1).
The antigen inactivated with 0.001 M BEI and concen-
trated by PEG precipitation method showed 2048 HA
titer, but the antigen inactivated by 0.3% formaldehyde
solution and concentrated by PEG precipitation method
did not show HA titer (Table 1). The concentrated an-
tigen suspended in PBS at pH 7.2 did not show HA ac-
tivity with goose erythrocytes, but the antigen sus-
pended in PBS at pH 9.0 showed 2048 HA titer (Table
2). The antigen by BEI inactivation method was titrated
with goose erythrocytes and showed 2048 HA titer. The
antigen prepared from suckling mice brain using su-
crose-acetone extraction method showed 256 HA titer
(Figure 1).
4. Discussion
Since children vaccination program was introduced in
Korea in 1960’s, JE vaccine has been inoculated into
most children. The JE vaccine program results in leading
low JE case in human [12]. However, the recent climate
change affects JEV infection rate in the human population
as 45 JEV cases were reported by the Korean Center for
Table 1. HA titer of JEV antigen according to inactivating
Supernatant Concentrated antigen
Inactivating agent Formaldehyde BEI* FormaldehydeBEI
HA titer <2 <2 <2 2048
*Binary ethylaminimine.
Table 2. Optimal buffer conditions for BEI method.
Concentrated antigen
Diluent Phosphate buffered
saline (PBS), pH 7.2
Borate buffered saline
(BBS), pH 9.2, BSA
HA titer <2 2048
Figure 1. Comparison between two JEV antigens by the HA
test. Each JEV antigens was diluted by serially two folds,
and the HA titer was expressed as the reciprocal of the
highest dilution of antigen showing complete hemagglutina-
tion. JEV antigen produced by PEG method represents
2048 HA titer and JEV antigen produced by sucrose ace-
tone method showed 256 HA titer.
Disease Control and Prevention between 2007 and 2010
[2]. On the other hand, veterinary authorities carried out
JEV serosurveillance in domestic animals including
swine, goats, cattle, horses and wild migrating birds. Es-
pecially, wild birds captured in South Korea showed
86.7% seropositive rate between 2007 and 2009 indicat-
ing that most wild birds captured in South Korea have
been exposed to JEV [12]. Therefore, it is necessary to
strengthen sero-survey of JE in several kinds of live-
In this study, we developed a new method to produce
JEV antigen without using suckling mice. During the
process of manufacturing JEV antigen, it is needed to
inactivate the antigen. Beta propiolactone, acetyleth-
lemine, formaldehyde and BEI have been used to inacti-
vate several kinds of virus in preparing inactivated vac-
cines [13,14]. The BEI acts on the nucleic acid of virus
and do not effect on the immunogenic properties of the
viral protein. In contrast, formaldehyde acts on the sur-
face protein of virus or bacteria and fixes the envelop
protein of JEV that plays a key role in agglutinating
goose erythrocytes and immunogenicity, tissue tropism,
cell fusion, infection and maturation [15]. Therefore, the
JEV antigen inactivated with 0.3% formaldehyde did not
react on hemagglutination.
Many kinds of vector-borne viruses can agglutinate a
broad range of animal species of erythrocytes, but, exact
experimental pH condition is required for each virus [16].
When the optimal pH for the JEV is deduced, further HA
titer will be carried out at the appropriate pH [17]. In this
study, BEI inactivated antigen suspended in PBS at pH
7.2 did not agglutinate goose erythrocytes, but the anti-
gen suspended in PBS at pH 9.0 containing bovine albu-
min revealed high HA activity with erythrocytes, indi-
cating that optimum buffer condition such as pH or BSA
should be applied for HA against JEV.
Sucrose acetone method using suckling mice has been
used to produce JEV antigen but this method provokes
animal welfare and environment contamination indicat-
ing that new method should be developed to replace the
method. In this study, HA titer of JEV antigen produced
by sucrose acetone method was found to be 256. Gener-
ally, the virus produced in newborn mice yields high titer,
but mouse brain material should be removed with several
treatments such as acetone [18,19]. The reason why the
antigen produced by sucrose acetone method show mod-
erate high HA activity may be assumed that the antigen
was extracted from brain homogenates inappropriately or
might be lost in the process of producing JEV antigen.
In conclusion, the present results suggested that BEI
inactivation method does not require treatment with ace-
tone and can thus reduce environmental contamination
with solvent. In addition, the JEV antigen inactivated by
BEI can reduce public health risks in carrying out HI test.
Copyright © 2012 SciRes. OJVM
Copyright © 2012 SciRes. OJVM
5. Acknowledgements
This work was financially supported by a grant (BAD14-
2011-12-02) from the Animal and Plant and Fisheries
Quarantine and Inspection Agency (QIA), Ministry for
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