Binary Ethylenimine Inactivated Japanese Encephalitis Virus Antigen Reveals Hemagglutination


Severe climate change and global warming may impact significantly on vector-borne disease including Japanese encephalitis (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 conventional 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 method 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.

Share and Cite:

D. Kun Yang, H. Hyun Kim, J. Ju Nah, K. Woo Lee and J. Young Song, "Binary Ethylenimine Inactivated Japanese Encephalitis Virus Antigen Reveals Hemagglutination," Open Journal of Veterinary Medicine, Vol. 2 No. 3, 2012, pp. 120-123. doi: 10.4236/ojvm.2012.23020.

Conflicts of Interest

The authors declare no conflicts of interest.


[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 Research Priorities,” Asia-Pacific Journal of Public Health, Vol. 23, No. 2, pp. 54-66, 2011.
[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, “Serosurveillance 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 Review,” 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. 2011, p. 472. doi:10.1186/1743-422X-8-472
[5] E. Konishi, Y. Sakai, Y. Kitai, A. Yamanaka, “Prevalence 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 Antibody 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, Including Hepatitis a Virus and Human Rotavirus, from Oyster, Water, and Sediment Samples,” Applied and Environmental Microbiology, Vol. 54, No. 8, 1988, pp. 1983-1988.
[10] D. H. Clarke and J. Casals, “Techniques for Hemagglutination and Hemagglutination-Inhibition with Arthropod-Borne Viruses,” American Journal of Tropical Medicine 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 Epidemiology 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 Application 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 Efficiently Internalized into Cultured Cells,” Vaccine, Vol. 29, No. 52, 2011, 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 Virology, Vol. 78, No. 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 Albopictus Cell Cultures,” American Journal of Tropical Medicine 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, “Immunological Equivalence between Mouse Brain-Derived and Vero Cell-Derived Japanese Encephalitis Vaccines,” Virus 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, “Recent Advancement in Flavivirus Vaccine Development,” Expert Reviews of Vaccines, Vol. 3, No. 2, 2004, pp. 199-220. doi:10.1586/14760584.3.2.199

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.