Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Study on Persistent Infection of Japanese Encephalitis Virus Beijing-l Strain in Serum-free Sf9 Cell Cultures

  • Kim, Hun (Green Cross Vaccine Corporation, and Department of Molecular Science and Technology, Ajou University) ;
  • Lee, Su-Jeen (Green Cross Vaccine Corporatio) ;
  • Park, Jin-Yong (Green Cross Vaccine Corporation, and Department of Molecular Science and Technology, Ajou University) ;
  • Park, Yong-Wook (Green Cross Vaccine Corporatio) ;
  • Kim, Hyun-Sung (Green Cross Vaccine Corporation, and Department of Biotechnology and Bioengineering. Inha University) ;
  • Kang, Heui-Yun (Department of Molecular Science and Technology, Ajou University) ;
  • Hur, Byung-Ki (Department of Biotechnology and Bioengineering. Inha University) ;
  • Ryu, Yeon-Woo (Department of Molecular Science and Technology, Ajou University) ;
  • Han, Sang-In (Department of Molecular Science and Technology, Ajou University)
  • Published : 2004.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8${\times}$l0$\^$6/ cells/$m\ell$ in a 500$m\ell$ spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-l strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15 % of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-l strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-l stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-l strain rose from 1.0${\times}$l0$\^$5/ to 1.5${\times}$l0$\^$6/ pfu/$m\ell$. The infected Sf9 cells could be subcultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-l strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-l strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.

Keywords

References

  1. Indian Med. Res. v.56 Establishment of carrier cultures of Aedes albopictus cell line infected with arboviruses Banerjee,K.;K.R.P.Singh
  2. Viroglogy v.21 Differences in maximum and minimum temperature among selected group A arboviruses Brown,A. https://doi.org/10.1016/0042-6822(63)90197-1
  3. Biolobicals v.24 Establishmen and characterization of Japanese B encephalitis virus persistent infection in the Sf9 insect cell line DaWeiFu;P.F.Zhang https://doi.org/10.1006/biol.1996.0031
  4. Curr.Top. Microbial.I mmunol. v.267 Japanese encephalitis virus:Ecology and epidemiology Endy,T.P.;A.Nisalak https://doi.org/10.1007/978-3-642-59403-8_2
  5. WO 97/04803 Method for industrially producing a japanese encephalitis vaccine, and resulting vaccine FangetBemard;FranconAlain;HeimendingerPierre
  6. Curr.Sci. v.72 no.12 Optimal pH conditions for the growth, release and stability of Japanese encephalitis virus in PS cell line Gangodkar,S.V.;S.P.Paranjape;V.D.Kadam;R.P.Deolankar
  7. Biken J. v.16 Growth of Japanese encephalitis virus inestablished lines of mosquito cells Igarashi,A.;F.Sasao;S.Wungkobkiat;K.Fukai
  8. J. Gen. Virol. v.40 Isolation of a Singhs Aedes albopictus cell clone sensitive to dengue and chikungunya viruses Igarashi,A. https://doi.org/10.1099/0022-1317-40-3-531
  9. EP 1 057 889 A1. 99923858 v.7 Enhanced immunogen for inactivated vaccine for infection with Japanese encephalitis viruses and process for producing the same IshikawaToyokazu;YoshiiHironori;ImagawaTadashi;IshibashiMasahide
  10. Am. J. Trop. Med. Hyg. v.18 Growth of Venezuelan and eastern equine encephalomyelitis viruses in tissue cultrues of minced Aedes aegypti larvae Johnson,J.W. https://doi.org/10.4269/ajtmh.1969.18.103
  11. Chin, J. Microbiol. Immunol. v.5 Study of infection of C6/36 cells with Japanse encephalitis virus Liang,X.J.;Y.M.Wang.
  12. In recombinant DNA technology and Application Cloning and expression of heterologous genes in insect cells with baculovirus vectors Luckow,V.A.
  13. Nat. Inst. Anim. Hlth. Quart. v.9 Growth characteristics and persistent infection of Japanese encephalitis virus in bovine kidney cell culture at different temperatures Morimoto,T.;T.Sugimor;Y.Miura;H.Sazawa;M.Watanabe
  14. In Vitro Establishment of persistent infections by flaviviruses in Aedes albopictus cell. In Invertebrate systems Ng.M.L.;E.G.Westaway
  15. J. Gen. Virol. v.71 Low pH-induced cell fusion in flavivirus type 2 in Aedes albopictus cell culture Randolph,V.B.;V.Stollar https://doi.org/10.1099/0022-1317-71-8-1845
  16. J. Gen. Virol. v.12 Replication of dengue virus type 2 in Aedes albopictus cell culture Sinarachatanant,P.;L.C.Olson
  17. Texas Agricultural Experiment Station and Texas A&M University College Station A manual of methods for baculovirus vectors and insect cell culture procedures Summers,M.D.;G.E.Smith
  18. The VAERS Working Group.Vaccine v.18 Adverse events after Japanese enccphalitis vaccination:review of postmarketing surveillance data from Japan and the United States Takahashi,H.;V.Pool;T.F.Tsail;R.T.Chen
  19. In vitro v.13 The establishment of two cell lines from the insect Spodoptera frugiperda Vaughn,J.L.;R.J.Goodwin;G.J.Tompkins;P.McCawley https://doi.org/10.1007/BF02615077
  20. 4th Asia-Pacific Biochemical Engineering Conference Study on the sensitivity of sf9 cells to the Japanese B encephalitis virus Wang,X.;QiYihua;OuyangFan.
  21. WHO. 1994. Japanese encephalitis. Wkly. epidemiol. rec. 69, 113-120 https://doi.org/10.1021/bp010120q
  22. Wkly. epidemiol. rec. v.69 Japancese encephalitis WHO
  23. Biotechnol.Progr v.18 Stationary and microcarrier cell culture processes for progpagating Japanese encephalitis virus Wu,S.C.;G.Y.hUANG https://doi.org/10.1021/bp010120q
  24. Am. J. Trop. Med. Hyg. v.17 Infection of grace's Antheraea cell with arboviruses Yunker,C.E.;J.Cory. https://doi.org/10.4269/ajtmh.1968.17.889
  25. J. Gen. Virol. v.74 St Louis encephalitis virus esablishes a productive, cytopathic and persistent infection of sf9 cells ZhangPeng-fei.;K.Michael;M.Jacqueline;J.M.Carol https://doi.org/10.1099/0022-1317-74-8-1703