Two Japanese encephalitis (JE) virus strains, namely a virulent JaOH0556 and an attenuated ML-17 derived from it were studied to investigate their in vitro phenotypic differences. When both strains were concentrated by polyethylene glycol (PEG) 6000 precipitation, infectivity of the ML-17 was recovered only 4.94% compared to 42.21% for the parental JaOH0566 strain, and the virion peak was very low for the ML-17 in contrast to a sharp peak for the JaOH0566 in sucrose density gradient centrifugation. When both strains were incubated with sucrose solutions, the ML-17 was slightly more labile than the JaOH0566 at high concentration of sucrose. These findings may indicate difference in the virion structure of both strains

Igarashi Akira

Morita Kouichi

Shameem Golam Masud Mohammad

Tanaka Mariko

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Trop. Med., 31 (2), 67- 74, June, 1989 67In Vitro Difference between an Attenuated ML-17and its Parental Virulent JaOH0566 Strainof Japanese Encephalitis VirusGolam Masud Mohammad SHAMEEM, Kouichi MORITA,Mariko TANAKA and Akira IGARASHIDepartment of Virology, Institute of Tropical Medicine,Nagasaki University, 12-4 Sakamoto-machi, Nagasaki 852, JapanAbstract: Two Japanese encephalitis (JE) virus strains, namely a virulent JaOH0556 andan attenuated ML-17 derived from it were studied to investigate their in vitrophenotypic differences. When both strains were concentrated by polyethylene glycol(PEG) 6000 precipitation, infectivity of the ML-17 was recovered only 4.94% comparedto 42.21% for the parental JaOH0566 strain, and the virion peak was very low for theML-17 in contrast to a sharp peak for the JaOH0566 in sucrose density gradient cen-trifugation. When both strains were incubated with sucrose solutions, the ML-17 wasslightly more labile than the JaOH0566 at high concentration of sucrose. These findingsmay indicate difference in the virion structure of both strains.Key words: Attenuation, Polyethylene glycol, Japanese encephalitis virusINTRODUCTIONJapanese encephalitis (JE) virus is a member of the family Flaviviridae (Westaway etal, 1985), and also a mosquito-borne arbovirus (Berge, 1975) with major vertebrateamplifier of swine (Scherer et al., 1959; Konno et al., 1966) and vector mosquitoes ofCulex tritaeniorhynchus (Mitamura et al, 1938; Buescher et al, 1959). JE has been countedamong important public health problems in East, Southeast and South Asia causing severeencephalitis in humans and horses with significant mortality and grave sequelae and alsoabortion of pregnant swine (Miles, 1960; Umenai et al, 1985; Monath, 1988). Althoughseveral strains isolated from different geographic areas showed different genomic struc-tures by RNA oligonucleotide fingerprint analysis (Hori, 1986), phenotypic and genotypicReceived for Publication, June 2, 1989.Contribution No. 2249 from the Institute of Tropical Medicine, Nagasaki University68differences between virulent and attenuated JE virus strains have not well beendocumented. An attenuated live vaccine strain, ML-17, was derived from a virulentJaOH0566 strain isolated from the brain of a fatal JE case through serial passages andrepeated plaque cloning (Yoshida et al, 1981). The vaccine strain snowed altered biologicalmarkers such as reduced virulence in experimental animals and reduced growth in Culextritaeniorhynchus, and was regarded as an appropriate live vaccine to immunize swine toprevent viremia and amplification of JE virus in nature･ However, this attenuated strainhas not been characterized for its in vitro markers. In order to analyze the ML-17 strain,repeated attempts to purify the virion could not yield sufficient amount of the purifiedvirion, although it showed similar growth pattern to its parental strain in cultured cells(Shameem βt aら1988). These findings suggested that the virion of ML-17 strain couldbe less stable than its parental strain･ In this paper, we describe that ML-17 strain ismore labile than its parental JaOHO566　strain for polyethylene glycol (PEG) treatmentwhich has been used routinely for virus purification.MATERIALS AND METHODSCelli．A clone C6／360f AedeS alb坤ictuS Cells（Igarashi，1978）was used for virus growth・The cells were gr0Wn at28℃in spinner b0ttlesin suspensi0n Withlliter／bottle（M0ritaandIgarashi，1989）with Eagle’s medium supplemented with O・2mM each of nonessentialamino acids（Eagle，1959）andlo％heat－inactivated fetalcalf serum（FCSつ．BHK21cellswere used for titration of the virusinfectivity and were grown at37℃　with the samemedium as C6／36cells・In the maintenance medium after virusinoculation，FCS concentra－ti0n WaS reduced t0　2％・昭rus：BothJaOH0566and ML・17strains were0btained fr0m KanonjiInstitute，ResearchFoundati0n for Micr0bialDiseases0f Osaka University（H0rietal．，1986），and seed viruseswere preparedin stationalculture of C6／36cells・1蒲mmqpemridaSe StainingqftheI｝iruS：Virusinfectivity was assayed byimmunoper0Ⅹidasestaining with slight modification from that of Okuno et aL，（1985）・Afterinoculati0n ofVirus specimens serially diluted by5％FCSin Eagle’s medium and2hr ads0rption at37℃，the cells0f96－WellmicroplateS Were C0Vered withl％Tragacanth gumin themaintenance medium andincubated at37℃in5％CO2for30hr．Then the cells were fix－ed by5％f0rmaldehydein ph0SPhate buffered saline（PBS），pH7・4，at ro0rh temperature（RT）f0r20min，and the cellmembrane was permealized byl％N0nidet P－40in PBSfor20min at RT．After reactions with anti－JE mouse serum and per0Ⅹidase－COnjugatedantトmouseIgG rabbitIgG（DAKO，Denmark），Peroxidase reacti0n WaS Carried out withl・2mg／mlof3，3’－diaminobezidine4HCIand o・01％H202in PBS at RT for20min．F0Ci0finfected cells were c0unted by stereosc0Pe andinfectivity was expressed as f0CuSforming unit（FFU）／ml．昭ruS C0nCentO雨ion andP）urification：The pr0Cedure was carried0ut aCCOrding to srivastava69et al, (1987). When C6/36 cells grew up to about 106 cells/ml, microcarrier (Cytodex-1,Pharmacia, Swedenつwas added to 2 mg/ml to attach the cells on it. After 12 hr, the cellson microcarrier were sedimented by standing the bottles on the bench and culture fluidwas removed by decantation･ Forty ml of seed virus was added to the sedimented cells ineach bottle and virus adsorption was carried out at RT for 2 hr. Then the cells were sup･plied by　750 ml/bottle of the maintenance medium and spinner culture was continued･After 2 days of virus inoculation, infected culture fluid was harvested and the cells weresupplied by fresh maintenance medium and infected fluid was harvested every alternativeday altogether 3 times･ Infected culture fluid (SI) was passed through No. 2 filter paper(Advantec‐Toyo, Japan), and centrifuged at 10,000g for 15 min at 4℃ to get the superna-tant (S2)･ Polyethylene glycol (PEG) 6000 (Wako Pure Chemical Industries, Japan) andNaCl were added to S2 to final concentrations of 6% and 0.5 M respectively. When theywere completely dissolvedーhe mixture was centrifuged at 10,000g for 30 min at 4℃ andthe supernatant (S3) was removed. The sediment was resuspended in STE buffer (0･1 MNad, 0･01 M Tris-HCl, 0･001 M EDTA, pH 7.4) using 24 ml for each harvest. Thesuspension (S4) was centrifuged at　20?000g for 15　min and the supernatant (S5) waslayered on 10 ml of 30-45% linear sucrose gradient and 2 ml of 15% sucrose in STE buf-fer･ The specimen was centrifuged at 20,000 rpm for 20 hr at 4℃ using SW28 rotor of aBeckman model L8-80M ultracentrifuge. Fractions were collected by ISCO density gra-dient fractionator model 640 recording OD254･Effec十of sucrose on the infectivity of JE vi･rus: The specimens S5 of both ML‐17 andJaOHO566 strains were diluted 1:10 with various concetration of sucrose solutions in STEbuffer to get the final sucrose concentration of O%, 15%,25%, 35% and 45%, and the mix-tures were incubated at 4℃ for 2 hr. Infectivity of these solutions was assayed as describ-ed above and compared with the titer of S5 which was diluted 1:10 by the virus diluentand incubated in parallel with the specimen of sucrose･RESULTSEffect of PEG on the infecti･vity of MLン17 and JaOHO566 strainsTable 1 shows the infectivity of both ML-17 and JaOHO566 strains at various stepsof virus purification･ Although virus titer was similar for both strains in the startingmaterial (SI) and ㎡ter filtration and clarification (S2), recovery of the infectivity in S4 was42･21% for JaOHO566 in contrast to 4･94% for the ML･17･ This difference cannot be ex-plained by poor precipitation of the ML-17 because the virus titer in S3 was 3.9% of theoriginal specimen for JaOHO566 in contrast to 0.03%　for ML‐17　strain･ The resultsrather suggest that the virion of ML‐17 is less stable than that of TaOHO566 for PEGtreatment･Figure 1 shows a sharp peak corresponding to the complete virion of the JaOHO566strain (upper panel) in contrast to a very low peak of the ML‐17 strain (lower panel) in70Table 1. Recovery of JaOH0566 and ML-17 strains by polyethylene glycol precipitationJaOH0566 ML-17Specimens * Totalinfe ctivityin FFURecoveryTotali nfe ctivityin FFURecoverySIS2S3S4S51 .40X1011 100.001.19X1011 85-005.46xl09 3.905.91X1010 42.215.48X1010 39.14S .OOxlO10 100.006.87xl010 85.872.15xl07 0.033.95X109 4.943.42X109 4.27* SI=Original infected fluids (750 ml); S2=Supernatant of high speed centrifugation after filtrationthrough filter paper; S3=Supernatant after PEG treatment of S2; S4=PEG precipitated S2 andresuspended in STE buffer (24 ml); S5=Supernatant of S4 after high speed centrifugation. Figuresrepresent average of 3 independent experiments.sucrose gradient sedimentation of specimen S5. The result may suggest that the virion ofML-17 was more easily disintegrated than that of JaOH0566 during sucrose gradientsedimentation.Effect of sucrose on the infectivity of ML-17 and JaOH0566 strainsIn order to test the possibility that ML-17 strain is more easily disintegrated thanits parental strain during sucrose gradient sedimentation, S5 specimens after PEG precipi-tion of both strains were incubated with various concentration of sucrose solutions at 4°Cfor 2 hr and the infectivity was titrated. Table 2 shows that incubation with sucrose solu-tions did not show very much apparent inactivation of the ML-17 than the JaOH0566,but at high concentration of sucrose (35% and 45%) recovery of the ML-17 was slightlyless than that of the JaOH0566. Both strains were inactivated a little in STE buffer com-pared with the original S5 specimen in virus diluent with 5% PCS in Eagle's medium.Table 2. Effect of sucrose on the infectivity of JaOH0566 and ML-17 strains after PEG treatmentJ aOH0566S ucroseConcentratio nin STE (%) I nfe ctivity Recoveryin FFU/ml %ML-17Infectivity Recoveryin FFU/ml %015253545l .GlxlO9 70.61.55X109 68.01.63xl09 71.51.69X109 74.11.85X109 81.10 .97X108 67.8l.llxlO8 77.61.15X108 80.40.87X108 60.80.93xl08 65.0O riginal* 2. 28x l09 100. 0 L43x l08 100.0* Specimen S5 before sucrose treatment and diluted with virus diluent instead of STE or sucrose solu-tions.711 .61.2Ià"§toCMà"soo0.400 .40JaOH0566ML-17D1 2 3 5 2125Fraction No.Fig. 1. Sucrose density gradient of JaOH0566 and ML-17 strains of Japanese encephalitis virus.Specimen S5 of the JaOH0566 (upper panel) or the ML-17 (lower panel) strain wasoverlayed on 10 ml of 30-45% linear sucrose gradient and 2 ml of 15% sucrose column andcentrifuged at 20,000 rpm for 20 hr at 4°C with SW28 rotor of a Beckman L8-80Multracentrifuge and fractions were collected by an ISCO density gradient fractionator model640 and OD254 was recorded. Volume of the fractions 1 to 4 was 6.0 ml and from 5 to 25was 0.6 ml. OD peak around fraction 21-23 corresponds to the complete virion.DISCUSSIONIn order to investigate the molecular basis of attenuation of the ML-17 strain,repeated attempts have been made in our laboratory to concentrate and purify this strainby PEG precipitation without successful recovery of the purified virion in quantity.Therefore, we tried to see the effect of PEG on the ML-17 strain as well as on itsparental JaOH0566 strain and found that the ML-17 was recovered much less than theJaOH0566 strain. From these data, together with the profile of sucrose density gradientsedimentation and the stability of the viral infectivity in sucrose solutions, we assume that72the virion of ML-17 strain was less stable than that of JaOH0566 strain, specially byPEG treatment. The results suggest that these 2 strains could have some differences-intheir virion structural proteins, which appears to be supported by the nucleotide sequenceanalysis on these 2 strains (Hori et al, 1988). Several investigators applied PEG precipita-tion for virus concentration, for example, plant viruses (Leberman et al, 1966), vesicularstomatitis virus (McSharry and Benzinger 1970), and bacteriophage (Yamamoto andAlberts 1970). In the previous experiment of virus growth and stability of several strainsof JE virus in virus diluent, the ML-17 and the JaOH0566 strains did not show anysignificant difference (Shameen et al, 1988). Therefore, reduced stability of the ML-17became apparent after it was treated with PEG. Hahn et al, (1987) compared nucleotidesequences of the virulent Asibi strain of yellow fever virus with the 17D vaccine strainderived from it. They argued that at least some of the difference in virulence between thetwo strains of a virus results from changes in the envelope protein that affect virus bin-ding to host receptors and such differences in receptor binding could result in the reducedneurotropism and vicerotropism exhibited by the vaccine strain. Our study is especially in-teresting because it represents some in vitro phenotypic difference between an attenuatedand its parental JE virus strains, which may be due to the mutation either in the struc-tural or nonstructural gene after passages in the tissue culture.ACKNOWLEDGEMENTSThe first author was sponsored by the Monbusho-Scholarship from the Ministry ofEducation, Science and Culture of Japan. The study was partly supported by aGrant-in-Aid for Scientific Research, No. 62480161 from the Ministry of Education,Science and Culture of Japan in the year of 1989.REFERENCES1) Berge, T. 0. (ed. ) (1975): International Catalogue of Arboviruses Including Certain Other Virusesof Vertebrates. 2nd ed. US Dept. Hlth. Educ. Welf. Publ. Hlth. Serv. Washington, D. C.2) Buescher, F. L., Scherer, W. F., Rosenberg, M. Z., Gresser, L, Hardy, J. L. & Bullock, H. R.(1959): Ecological studies of Japanese encephalitis in Japan. II. Mosquito infection. Amer. J.Trop. Med. Hyg., 8, 651-664.3) Eagle, H. (1959): Amino acid metabolism in mammalian cell cultures. Science, 130, 432-437.4) Hahn, C. S., Dalrymple, J. M., Strauss, J. H. & Rice, C. M. (1987): Comparison of the virulentAsibi strain of yellow fever virus with the 17D vaccine strain derived from it. Proc. Natl. Acad.Sci. USA., 84, 2019-2023.5) Hori, H. (1986): Oligonucleotide fingerprint analysis on Japanese encephalitis (JE) virus strains ofdifferent geographic origins. Trop. Med., 28, 179-190.736) Hori, H., Igarashi, A., Yoshida, I. & Takagi, M. (1986): Oligonucleotide fingerprint analysis ofJapanese encephalitis virus strains after passage histories. Acta Virol., 30, 428-431.7) Hori, H., Tanaka, M., Fuke, L, Haishi, S. & Igarashi, A. (1988): Analysis on an attenuatedJapanese encephalitis virus strain at the molecular level. Abstract of the 36th Annual Meeting ofJapanese Virologists Society, 419, Tokyo.8) Igarashi, A. (1978): Isolation of a Singh's Aedes albopictus cell clone sensitive to dengue andchikungunya viruses. J. Gen. Virol., 40, 531-544.9) Konno, J., Endo. K., Agatsuma, H. & Ishida, N. (1966): Cyclic outbreaks of Japanese encephalitisamong pigs and humans. Amer. J. Epidemiol., 84, 292-300.10) Leberman, R. (1966): The isolation of plant viruses by means of "simple" coacervates. Virology,30, 341-347.ll) McSharry, J. & Benzinger, R. (1970): Concentration and purification of vesicular stomatitis virusby polyethylene glycol "precipitation". Virology, 40, 745-746.12) Miles, J. A. R. (1960): Epidemiology of arthropod-borne encephalitides. Bull. WHO., 22,339-371.13) Mitamura, A., Kitaoka, M., Mori, K. & Ohkubo, K. (1938): Demonstration of Japaneseencephalitis virus in natural mosquitoes- An evidence of disease transmission by mosquitoes.Ninon-Ijishinshi, 62, 820-824.14) Monath, T. P. (1988): Japanese encephalitis - a plague of the orient. N. Engl. J. Med., 319,641-643.15) Morita, K. & Igarashi, A. (1989): Suspension culture of Aedes albopictus cells for flavivirus massproduction. J. Tissue Culture Methods; in press.16) Okuno, Y., Fukunaga, T., Tadano, M., Okamoto, Y., Ohnishi, T. & Takagi, M. (1985): Rapidfocus neutralization test of Japanese encephalitis virus in microtiter system. Arch. Virol., 86,129-135.17) Scherer, W. R, Moyer, J. T., Izumi, T., Gresser, I. & McCown, J. (1959): Ecological studies ofJapanese encephalitis virus in Japan. IV. Swine infection. Amer. J. Trop. Med. Hyg., 8,698-706.18) Shameem, G. M. M., Morita, K. & Igarashi, A. (1988): Growth patterns of six strains ofJapanese encephalitis virus. Trop Med., 30, 233-238.19) Srivastava, A. K., Aira, Y., Mori, C., Kobayashi, Y. & Igarashi, A. (1987): Antigenicity ofJapanese encephalitis virus envelope glycoprotein V3 (E) and its cyanogen bromide cleavedfragments examined by monoclonal antibodied and Western blotting. Arch. Virol., 96, 97-107.20) Umenai, T., Krzysko, R., Bektimirov, A. & Assaad, F. A. (1985): Japanese encephalitis: currentworldwide status. Bull. WHO., 63, 625-631.21) 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, 183-192.22) Yamamoto, K. R. & Alberts, B. M. (1970): Rapid bacteriophage sedimentation in the presence ofpolyethylene glycol and its application to large-scale virus purification. Virology, 40 734-744.7423) Yoshida, L, Takagi, M., Inokuma, E., Goda, H., Ono, K., Takaku, K. & Oku, J. (1981): Establish-ment of an attenuated ML-17 strain of Japanese encephalitis virus. Biken J., 24, 47-67.

In Vitro Difference between an Attenuated ML-17 and its Parental Virulent JaOH0566 Strain of Japanese Encephalitis Virus

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In Vitro Difference between an Attenuated ML-17 and its Parental Virulent JaOH0566 Strain of Japanese Encephalitis Virus

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