Growths of 6 strains of Japanese encephalitis (JE) virus were studied in mosquito (C6/36) and baby hamster kidney (BHK21) cell cultures at 4 different temperatures. At 28℃, 3 strains (Nakayama, Beijing-1, JaOArS982) grew slower than other 3 strains (JaGAr01, JaOH0566, ML17) in C6/36 cells although their infectivity increased from 1 day after infection. On the other hand, infectivity of all strains increased from 2 days after infection in BHK21 cells. At 33 and 37℃, growth of JaOArS982 strain in C6/36 cells was shut-off 2 and 1 day after infection, respectively, while growths of all strains in BHK21 cells were shut-off 2 days after infection. At 39℃, growths of all strains were shut-off 1 day after infection in both cell lines, and infectivity of Nakayama strain showed little increase in C6/36 cells. The results indicated some host cell factor(s) regulating replication of JE virus, especially in C6/36

Igarashi Akira

Morita Kouichi

Shameem Golam Masud Mohammad

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T rop. Med., 30 (4), 233-238, December, 1988 233Growth Patterns of Six Strains ofJapanese Encephalitis VirusGolam Masud Mohammad SHAMEEM,Kouichi MORITAand Akira IGARASHIDepartment of Virology, Institute of Tropical Medicine,Nagasaki University, 12-4 Sakamoto-machi, Nagasaki 852, JapanAbstract: Growths of 6 strains of Japanese encephalitis (JE) virus were studied in mos-quito (C6/36) and baby hamster kidney (BHK21) cell cultures at 4 different temperatures.At 28•Ž, 3 strains (Nakayama, Beijing-1, JaOArS982) grew slower than other 3 strains(JaGAr01, JaOH0566, ML17) in C6/36 cells although their infectivity increased from 1day after infection. On the other hand, infectivity of all strains increased from 2 daysafter infection in BHK21 cells. At 33 and 37•Ž, growth of JaOArS982 strain in C6/36cells was shut-off 2 and 1 day after infection, respectively, while growths of all strains inBHK21 cells were shut-off 2 days after infection. At 39•Ž, growths of all strains wereshut-off 1 day after infection in both cell lines, and infectivity of Nakayama strain show-ed little increase in C6/36 cells. The results indicated some host cell factor(s) regulatingreplication of JE virus, especially in C6/36.Key words: Growth pattern, Japanese encephalitis, InfectivityINTRODUCTIONJapanese encephalitis (JE) virus is a member of the family Flaviviridae (Westaway etal, 1985), which was formerly classified as mosquito-borne group B arbovirus (Clarke andCasals, 1965; Fiedman, 1968). JE is a serious public health problem with significant mortali-ty and grave sequelae in many Asian countries (Miles, 1960; Umenai et al, 1985). Sincethe first isolation in 1935 in Japan, many strains of JE virus have been isolated from mos-quitoes, birds, swine, horses, and humen. In spite of many investigations of differentaspects of JE virus, detailed knowledge on its growth patterns in mosquito and mam-malian cell lines using different strains at various temperatures is rather limited.This paper describes growth patterns of 6 strains of JE virus at 4 temperatures inorder to pinpoint the host-virus-temperature relationship in JE virus production.Received for Publication, October 20, "1988.Contribution No. 2128 from the Institute of Tropical Medicine, Nagasaki University.234MATERIALS AND METHODSCell: Aedes albopictus, clone C6/36 cells (Igarashi, 1978) were grown at 28°C with cellgrowth medium of Eagle's minimal essential medium supplemented with 0.2mM each ofnon-essential amino acids (Eagle, 1959) and 10% heat-inactivated fetal calf serum (FCS).BHK21 cells were grown with the same medium but at 37°C.Virus: Six strains of JE virus were used in this study: Nakayama Yoken, Beijing-1,JaGArOl, JaOH0566, ML17 and JaOArS982, and their origins were described previously(Hori et al, 1986a; b). Con fluent cultures of C6/36 cells in 750cm3 Roux bottles were in-oculated with each strain of JE virus. After 2 hours of adsorption, the cells were fed with40 ml/bottle of maintenance medium (cell growth medium from which FCS concentrationwas reduced to 2%) and incubated at 28°C. After 3 days, infected culture fluids wereharvested and stored in aliquots at -70°C as seed virus and their infectivities were deter-mined.Virus growth experiment: Growth medium was removed by aspiration from C6/36 orBHK21 cells on 24 well semimicroplates (Falcon 3047, USA), and O.lml of seed virus wasinoculated into each well at appropriate dilution to input multiplicity of infection of 0.1PFU/cell. Virus adsorption was carried out for 2 hours with spreading the inoculum every30 minutes. Then, residual virus was removed and 1ml of maintenance medium was addedto each well and the cells were incubated in 5% C02~atmosphere at respectivetemperatures. Aliquots of infected culture fluid was harvested every day from duplicatewells and virus infectivity in pooled specimens was titrated.Infectivity titration of the virus: The method was modifed from that described byHashimoto et al. (1971). Specimens were serially diluted in 10-fold steps with 5% FCS inEagle's medium and inoculated to BHK21 cells on semimicroplates after their growthmedium was removed, using 0.1 ml/well and duplicate wells for each dilution. After 2hours of adsorption at 37°C by spreading the inoculum every 30 minutes, residual viruswas removed and the cells were covered with 1 ml/well of overlay medium (1.5% methylcellulose and \% FCS in Eagle's medium) and incubated at 37°C in 5% C02-atmospherefor 5 days. The overlay medium was removed by rinsing with phosphate buffered salinewith calcium and magnesium. The cells were fixed with cold methanol for 30 minutes andstained with 0.1% Trypan blue at room temperature for 1 hour to reveal the plaques. In-fectivity of the virus was recorded as plaque forming unit (PFU) per ml.RESULTSThe growth curve of 6 different strains of JE virus in C6/36 cells and BHK21 cellsat 4 different temperatures were shown in Fig. 1. At 28°C, 3 strains of JE virus(Nakayama, Beijing- 1, JaOArS982) grew slower than 3 other strains (JaGArOl, JaOH0566,ML17) in C6/36 cells, and the difference was remarkable 1 day after infection, when all2358.à"-^^Ax°^^S>5^ . B^"----r^A /à" /\f8765EDL-~ 8D)_O7A#£ =*:Io^1^à"'-fcC '^à"^^^-m D/à"^^^S?^"/f£^-\ ^* -I/IsfVoA4O x<y_ /'/'I'I,' >3\V' B.* 'AEIs*6543*FV)HIPVVa^' v*sw0 3 4 0Days of IncubationGrowth curve of 6 strains of JE virus in C6/36 cells and BHK21 cells at 4temperatures.Each of the 6 strains of JE virus, Nakayama (A A), Beijing-1 (à" à"),JaGAOl (D D), JaOH0566 (A A), ML17 (à" à"), and JaOArS982à"iO O), was inoculated to C6/36 (A, C, E, and G) or BHK21 (B, D, F, and H)cells, and incubated at 28°C (A and B), 33°C (C and D), 37°C (E and F), and 39°C(G and H), to assay virus infectivity in the infected fluids by plaque titrtion onBHK21 cells at 37°C. Infectivity was undetectable for Nakayama, Beijing-1,JaGArOl, and JaOArS982 strains 4 days after infection in C6/36 cells at 39°C (G),for Nakayama, JaGArOl, JaOH0566 and ML17 strains 3 days after infection, andfor all strains 4 days after infection in BHK21 cells at 39°C (H).236strains showed titer increase. While in BHK21 cells almost no titer increase was observed 1 dayafter infection for all strains, and their titers increased 2-3 days after infection. Dif-ference among virus titers of various strains in BHK21 cells 3 days after infection was notso remarkable, although JaGArOl showed the highest and Nagayama the lowest titer,respectively, 2 days after infection.At 33°C, all strains grew up faster than at 28°C both in C6/36 cells and BHK21 cellswith similar rates up to 2 days after infection. But after 2 days of incubation, virus produc-tion was shut-off for JaOArS982 strain in C6/36 cells and all strains in BHK21 cells, andinfectivity of 5 remaining strains in C6/36 cells did not change significantly.Similar findings were obtained at 37°C, but shut-off of JaOArS982 strain in C6/36cells took place earlier, that is 1 day after infection.At 39°C, all strains showed sharp decline in their titers after 1 day's increase in bothcell lines, and infectivity of Nakayama strain increased less than other strains in C6/36cells. Such strain difference was not observed in infected BHK21 cells.A87c;x^*\\E:DU_Q_à"^sCOo65432\ \s \\ \x\\\&\\v}>0I/. x\V\M\*\\\\ \\\\\\v\ \\\**\\'%\ \V-AV'%x\^\\ \\ \\ \\ \\\\\s\B12 3 0 1Days of incubationFig. 2. Heat-stability of JE virus infactivityEach of the 6 strains of JE virus, Nakayama (A A), Beijing-1 (à" à"),JaGAOl (D D), JaOH0566 (A A), ML17 (à" à"). and JaOArS982(O O), was inoculated at 37°C (A), and 39°C (B), and specimens were sampledevery day to assay their infectivities on BHK21 cells at 37°C. Infectivity was undetec-table for Nakayama and Beijing-1 strains 3 days after incubation at 37°C (A), and forall strains 3 days after incubation at 39°C (B).237In order to explain the different growth curves for 6 strains of JE virus, heat-stabili-ty experiments were carried out. Each strain of seed virus was incubated at 37 and 39°Cand specimens were titrated for their infectivity every day until 4 days of incubation. Theresults in Fig. 2 showed that at 37°C infectivity of all strains decreased 2-3 logs within aday, and Nakayama and Beijing- 1 strains were more rapidly inactivated than other strainsafter 2 days of incubation and their infectivities became undetectable on the 3rd day.While, other strains were inactivated slower after 2 days of incubation and low levels of in-fectivities were still detectable evan after 3 days of incubation. After 4 days of incubation,infectivity was not detectable for all strains.At 39°C, inactivation rate was almost similar to that at 37°C for the 1st day of in-cubation and Nakayama and Beijing-1 strains were slightly more rapidly inactivated thanother strains during subsequent days of incubation. Infectivity became undetectable after 3days of incubation for all strains.DISCUSSIONRecently Igarashi and Srivastava (1988) reported that growth of some flaviviruses isdependent on the combination of virus and host cells. In the present study, we foundsome difference in the growth patterns of JE virus strains, particularly in C6/36 cells,although their basic growth patterns were essentially similar to all the strains in BHK21cells. Since no significant difference was observed among JE virus strains in their heat-stability and all strains showed similar growth rates in BHK21 cells, the shut-off ofJaOArS982 strain in C6/36 cells at 33 and 37°C after initial growth phase cannot be explain-ed by its reduced heat-stability. Therefore, the shut-off phenomenon could be the result ofreduced virus production, either by regulation on the processes of viral RNA replication,assembly and maturation which depend on some host cell factor(s), or by viral cytopathiceffect which significantly damages host macromolecular syntheses required for the viralproduction. These possibilities should be investigated by further study.ML17 is an attenuated vaccine strain for swine and was obtained from JaOH0566strain by serial passages and plaque purification in cell cultures at reduced temperatures,and has altered biological characteristics such as reduced swine viremia, reduced mouseneurovirulence, and reduced infectivity to vector mosquitoes, Culex tritaeniorhynchus(Yoshida et at, 1981). It is therefore unexpected that no significant difference was foundbetween JaOH0566 and ML17 strains in their growth patterns in C6/36 cells and BHK21cells at different temperatures. The result shows that in vitro markers do not alwaysparallel to in vivo markers.ACKNOWLEDGEMENTSThe first author was supported by Monbusho-scholarship, and the study was partlysupported by the Grant in Aid for Scientific research, No. 62480161 from the Ministry ofEducation, Science and Culture of Japan in the year of 1988.238REFERENCES1) Clarke, D. H. & Casals, J. (1965): Arboviruses; group B. pp606-658. In F. L. Horsfall, Jr. & I.Tamm (ed.). Viral and Rickettsial Infections of man. J. B. Lippincott, Philadelphia & Montreal.2) Eagle, H. (1959) Amino acid metabolism in mamalian cell cultures. Science, 130, 432-437.3) Friedman, R. M. (1968): Structural and non structural proteins of an arbovirus. J. Virol., 2, 1076-1080.4) Hashimoto, N., Yamada, K. & Kanamitsu, M. (1971): A microtiter method for assay of neutralis-ing antibodies against group B arboviruses. Virus, 21, 55-59.5) Hori, H., Morita, K. & Igarashi, A. (1986a): Oligonucleotide fingerprint analysis on Japaneseencephalitis virus strains isolated in Japan and Thailand. Acta Virol., 30, 353-359.6) Hori, H., Igarashi, A., Yoshida, I., & Takagi, M. (1986b): Oligonucleotide fingerprint analysis onJapanese encephalitis virus strains after passage histories. Acta Virol., 30, 428-431.7) Igarashi, A. (1978): Isolation of a Singh's Aedes albopictus cell clone sensitive to dengue andchikungunya viruses. J. Gen. Virol., 40, 531-544.8) Igarashi, A. & Srivastava, A. K. (1988): suspension culture of Aedes albopictus, clone C6/36, cellsand its application to the production of flaviviruses. pp!37-139. In Y. Kuroda, E. Kurstak & K.Maramorosch (ed.). Invertebrate and Fish Tissue Culture. Japan Scientific Socities Press, Tokyo /Springer-Verlag, Berlin.9) Miles, J. A. R. (1960): Epidemiology of arthropod-borne encephalitides. Bull. WHO., 22, 339-37110) Umenai, T., Krzysko, R., Bektimirov, A. & Assaad, F. A. (1985): Japanese encephalitis: currentworld wide status. Bull. WHO., 63, 625-631.ll) Westaway, E. G., Brinton, M. A., Gaidamovich, S. Ya., Horzinek, M. C., Igarashi, A.,Kaariainen, L., Lvov, D. K., Porterfield, J. S., Russell, P. K. & Trent, D. W. (1985):Flaviviridae. Intervirology, 24, 183- 192.12) Yoshida, L, Takagi, M., Inokuma, E., Goda, H., Ono, K., Takaku, K. & Oka, J. (1981): Establish-ment of an attenuated ML17 strain of Japanese encephalitis virus. Biken J., 24, 47-67.

Growth Patterns of Six Strains of Japanese Encephalitis Virus

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Growth Patterns of Six Strains of Japanese Encephalitis Virus

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