Binary Ethylenimine Inactivated Japanese Encephalitis Virus Antigen Reveals Hemagglutination

doi:10.4236/ojvm.2012.23020

Paper Menu >>

Journal Menu >>

Open Journal of Veterinary Medicine, 2012, 2, 120-123

http://dx.doi.org/10.4236/ojvm.2012.23020 Published Online September 2012 (http://www.SciRP.org/journal/ojvm)

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: *[email protected]

Received June 28, 2012; revised July 23, 2012; accepted July 31, 2012

ABSTRACT

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.

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

D.-K. YANG ET AL.

122

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

agents.

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-

stock.

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.

D.-K. YANG ET AL.

123

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

Food, Agriculture, Forestry and Fisheries (MIFAFF),

Korea.

REFERENCES

[1] D. Harley, P. Bi, G. Hall, A. Swaminathan, S. Tong and

C. Williams, “Climate Change and Infectious Diseases in

Australia: Future Prospects, Adaptation Options, and Re-

search Priorities,” Asia-Pacific Journal of Public Health,

Vol. 23, No. 2, 2011, pp. 54-66.

[2] D. K. Yang, Y. I. Oh, H. R. Kim, Y. J. Lee, O. K. Moon,

H. Yoon, B. Kim, K. W. Lee and J. Y. Song, “Serosur-

veillance for Japanese Encephalitis Virus in Wild Birds

Captured in Korea,” Journal of Veterinary Science, Vol.

12, No. 4, 2011, pp. 373-377.

doi:10.4142/jvs.2011.12.4.373

[3] G. L. Campbell, S. L. Hills, M. Fischer, J. A. Jacobson, C.

H. Hoke, J. M. Hombach, A. A. Marfin, T. Solomon, T. F.

Tsai, V. D. Tsu and A. S. Ginsburg, “Estimated Global

Incidence of Japanese Encephalitis: A Systematic Re-

view,” Bull World Health Organization, Vol. 89, No. 10,

2011, pp. 766-774, doi:10.2471/BLT.10.085233

[4] Q. S. Cao, X. M. Li, Q. Y. Zhu, D. D. Wang, H. C. Chen

and P. Qian, “Isolation and Molecular Characterization of

Genotype 1 Japanese Encephalitis Virus SX09S-01, from

Pigs in China,” Virology Journal, Vol. 8, No. 1, 2011, p.

472. doi:10.1186/1743-422X-8-472

[5] E. Konishi, Y. Sakai, Y. Kitai and A. Yamanaka, “Preva-

lence of Antibodies to Japanese Encephalitis Virus among

Inhabitants in Java Island, Indonesia, with a Small Pig

Population,” American Journal of Tropical Medicine and

Hygiene, Vol. 80, No. 5, 2009, pp. 856-861.

[6] E. Konishi, M. Shoda and T. Kondo, “Prevalence of An-

tibody to Japanese Encephalitis Virus Nonstructural 1

Protein among Racehorses in Japan: Indication of Natural

Infection and Need for Continuous Vaccination,” Vaccine,

Vol. 22, No. 9-10, 2004, pp. 1097-1103.

doi:10.1016/j.vaccine.2003.10.001

[7] O. P. Larghi and A. E. Nebel, “Rabies Virus Inactivation

by Binary Ethylenimine: New Method for Inactivated

Vaccine Production,” Journal of Clinical Microbiology,

Vol. 11, No. 2, 1980, pp. 120-122.

[8] R. Putnak, D. A. Barvir, J. M. Burrous, D. R. Dubois, V.

M. D’Andrea, C. H. Hoke, J. C. Sadoff and K. H. Eckels,

“Development of a Purified, Inactivated, Dengue-2 Virus

Vaccine Prototype in Vero Cells: Immunogenicity and

Protection in Mice and Rhesus Monkeys,” Journal of

Infectious Diseases, Vol. 174, No. 6, 1996, pp. 1176-1184.

doi:10.1093/infdis/174.6.1176

[9] G. D. Lewis and T. G. Metcalf, “Polyethylene Glycol

Precipitation for Recovery of Pathogenic Viruses, In-

cluding Hepatitis a Virus and Human Rotavirus, from

Oyster, Water, and Sediment Samples,” Applied and En-

vironmental Microbiology, Vol. 54, No. 8, 1988, pp.

1983-1988.

[10] D. H. Clarke and J. Casals, “Techniques for Hemaggluti-

nation and Hemagglutination-Inhibition with Arthro-

pod-Borne Viruses,” American Journal of Tropical Medi-

cine and Hygiene, Vol. 7, No. 5, 1958, pp. 561-573.

[11] D. K. Yang, B. H. Kim, C. H. Kweon, J. H. Kwon, S. I.

Lim and H. R. Han, “Biophysical Characterization of

Japanese Encephalitis Virus (KV1899) Isolated from Pigs

in Korea,” Journal of Veterinary Science, Vol. 5, No. 2,

2004, pp. 125-130.

[12] S. M. Yun, J. E. Cho, Y. R. Ju, S. Y. Kim, J. Ryou, M. G.

Han, W. Y. Choi and Y. E. Jeong, “Molecular Epidemio-

logy of Japanese Encephalitis Virus Circulating in South

Korea, 1983-2005,” Virology Journal, Vol. 7, No. 14,

2010, p. 127. doi:10.1186/1743-422X-7-127

[13] H. G. Bahnemann, “Inactivation of Viral Antigens for

Vaccine Preparation with Particular Reference to the Ap-

plication of Binary Ethylenimine,” Vaccine, Vol. 8, No. 4,

1990, pp. 299-303. doi:10.1016/0264-410X(90)90083-X

[14] M. A. Martín-Acebes, A. Vázquez-Calvo, M. González-

Magaldi and F. Sobrino, “Foot-and-Mouth Disease Virus

Particles Inactivated with Binary Ethylenimine Are Effi-

ciently Internalized into Cultured Cells,” Vaccine, Vol. 29,

No. 52, 2011, pp. 9655-9562.

doi:10.1016/j.vaccine.2011.10.031

[15] P. C. McMinn, “The Molecular Basis of Virulence of the

Encephalitogenic Flaviviruses,” Journal of General Vi-

rology, Vol. 78, No. 11, 1997, pp. 2711-2722.

[16] E. N. Meeusen, J. Walker, A. Peters, P. P. Pastoret and G.

Jungersen, “Current Status of Veterinary Vaccines,”

Clinical Microbiology Reviews, Vol. 20, No. 3, 2007, pp.

489-510. doi:10.1128/CMR.00005-07

[17] A. Igarashi, K. Buei, N. Ueba, M. Yoshida, S. Ito, H.

Nakamura, F. Sasao and K. Fukai, “Isolation of Viruses

from Female Culex Tritaeniorhynchusin Aedes Albopic-

tus Cell Cultures,” American Journal of Tropical Medi-

cine and Hygiene, Vol. 30, No. 2, 1981, pp. 449-460.

[18] M. Abe, K. Shiosaki, L. Hammar, K. Sonoda, L. Xing, S.

Kuzuhara, Y. Kino and R. Holland Cheng, “Immu-

nological Equivalence between Mouse Brain-Derived and

Vero Cell-Derived Japanese Encephalitis Vaccines,” Vi-

rus Research, Vol. 121, No. 2, 2006, pp. 152-160.

doi:10.1016/j.virusres.2006.05.004

[19] G. J. Chang, G. Kuno, D. E. Purdy and B. S. Davis, “Re-

cent Advancement in Flavivirus Vaccine Development,”

Expert Reviews of Vaccines, Vol. 3, No. 2, 2004, pp. 199-

220. doi:10.1586/14760584.3.2.199