Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures.
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 x 10(6) cells/ml in a 500ml 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-1 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-1 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-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml. The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 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.
Kim
Hun
H
Green Cross Vaccine Corporation, 227-3 Kugal-Ri, Kiheung-Eup, Yongin 449-903, Korea. hkim@greencrossvaccine.com
Lee
Su Jeen
SJ
Park
Jin Yong
JY
Park
Yong Wook
YW
Kim
Hyun Sung
HS
Kang
Heui-Yun
HY
Hur
Byung-Ki
BK
Ryu
Yeon-Woo
YW
Han
Sang In
SI
Kim
Jong Su
JS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D003470:Culture Media / Q000737:chemistry; D016895:Culture Media, Serum-Free; D003588:Cytopathogenic Effect, Viral; D003594:Cytoplasmic Granules; D004664:Encephalitis Virus, Japanese / Q000254:growth & development* / Q000302:isolation & purification* / Q000648:ultrastructure; D015726:Giant Cells; D013194:Staining and Labeling; D014461:Ultracentrifugation; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000032:analysis / Q000276:immunology; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000032:analysis / Q000276:immunology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D003470:Culture Media; D016895:Culture Media, Serum-Free; D014759:Viral Envelope Proteins; D014764:Viral Proteins