Respiratory syncytial (RS) virus can be purified without losing its infectivity provided that each step of purification is carried out using NT buffer containing over 20% sucrose. Firstly, the virus grown on HES cells is efficiently removed from the culture fluid by precipitating with polyethylene glycol (PEG) 6,000, and the precipitate is suspended in a small amount of 20% sucrose-NT buffer, which results in about a 24-fold concentration of the original material. Then this suspension is centrifugated through 30% sucrose-NT buffer to obtain pellets, which are again suspended in 20% sucrose-NT buffer. This suspension is further centrifuged by discontinuous and linear sucrose density gradient. Finally, the specific infectivity of the purified virus was increased about 3,000-fold over that of the original material.</p

Ueba, Osamu

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Acta Medica OkayamaVolume 32, Issue 4 1978 Article 2AUGUST 1978Respiratory syncytial virus. I. Concentrationand purification of the infectious virusOsamu Ueba∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Respiratory syncytial virus. I. Concentrationand purification of the infectious virus∗Osamu UebaAbstractRespiratory syncytial (RS) virus can be purified without losing its infectivity provided thateach step of purification is carried out using NT buffer containing over 20% sucrose. Firstly,the virus grown on HES cells is efficiently removed from the culture fluid by precipitating withpolyethylene glycol (PEG) 6,000, and the precipitate is suspended in a small amount of 20%sucrose-NT buffer, which results in about a 24-fold concentration of the original material. Thenthis suspension is centrifugated through 30% sucrose-NT buffer to obtain pellets, which are againsuspended in 20% sucrose-NT buffer. This suspension is further centrifuged by discontinuous andlinear sucrose density gradient. Finally, the specific infectivity of the purified virus was increasedabout 3,000-fold over that of the original material.KEYWORDS: respiratory syncytial virus, purification∗PMID: 153087 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 32, (4), 265-272 (1978)RESPIRATORY SYNCYTIAL VIRUSI. CONCENTRATION AND PURIFICATION OFTHE INFECTIOUS VIRUSOsamu UEBADepartment of Virology, Okayama University Medical School,Okayama 700, Japan (Director .. Prof. J. Tawara)Received March 23, 1978Abstract. Respiratory syncytial (RS) virus can be purified withoutlosing its infectivity provided that each step of purification is carried outusing NT buffer containing over 20% sucrose. Firstly, the virus grownon HES cells is efficiently removed from the culture fluid by precipitat-ing with polyethylene glycol (PEG) 6,000, and the precipitate is sus-pended in a small amount of 20% sucrose-NT buffer, which results inabout a 24-fold concentration of the original material. Then this sus-pension is centrifuged through 30% sucrose-NT buffer to obtain pellets,which are again suspended in 20% sucrose-NT buffer. This suspensionis further centrifuged by discontinuous and linear sucrose density gradi-ent. Finally, the specific infectivity of the purified virus was increasedabout 3,000-fold over that of the original material.Key words: respiratory syncytial virus, purificationRS virus is a virus that induces very severe respiratory disease in infants (1).This virus, which in many respects resembles the paramyxoviruses (2-4), isclassified in the paramyxoviridae family (5). However, it has not been fullycharacterised because of the size of its nucleocapsid (6-8), its inability to causehemagglutination (9), its lack of neuraminidase activity (10) as well as thedifference in the known polypeptide components (11)"Such points will become clearer with further study of its biochemicalcharacteristics and morphogenesis. The reason for the lack of progress in thisfield of study is that it is very difficult to concentrate and purify intact RS virusbecause of its instability. Recently, Wunner and Pringle (11) and Levine (12)obtained partially purified virus and reported on the viral protein thereof. Inboth cases, the study was conducted with a small amount of isotopically labeledvirus. For examination of the relationship between viral protein and its bio-logical activities, large amounts of purified virus are required. So it is necessaryto develop a more suitable method for the concentration and purification of largeamounts of the virus.RS virus is sensitive to changes in the temperature and pH as well as to thefreezing-thawing (13). Its infectivity is also markedly reduced in water and2651Ueba: Respiratory syncytial virus. I. Concentration and purification ofProduced by The Berkeley Electronic Press, 1978266 O. UEBAbuffer solution (14,15). However, fairly effective stabilizers have been found.Inorganic salts such as 1M NaCl, 1M MgS04, 1M NaZS04' and 1M glucose or1M sorbitol are good stabilizers of RS virus. It is also known that this virus canbe kept stable in 44.5% sucrose solution at 4°C and --70°C for a long period oftime (16, 17).Therefore, the author studied the stabilizing conditions of sucrose suited tothe medium used for the purification of RS virus and methods of concentrationand purification.MATERIALS AND METHODSVirus. The long strain of RS virus used was donated by Dr. Reisaku Konoof the National Institute of Health, Tokyo. Stock virus was prepared in HEScell cultures.Cell culture. HES cells, derived from human embryonic skin and establishedin our laboratory, were cultured in a roller bottle. The cells were grown inEagle's minimal essential medium (MEM) supplemented with 3% fetal calfserum (FCS), and the infected cells were maintained in MEM containing 2%FCS.Virus survival experiment. Sucrose solutions of 10%, 15%, 20%, 30%, 40%(w/w) were prepared in NT buffer solution composed of 0.15 M NaCl and 0.05 MTris (hydroxyl methyl) aminomethane-chloride (pH 7.5). The stock virus wasdiluted with NT buffer and sucrose solution of each concentration in the ratio of1 : 30, and left standing at 4°C. Thereafter the infectivity titer was determinedevery hour.Virus propagation. HES cells were infected at a multiplicity of approximately1 PFUjcell. After 2 h of adsorption at 33°C, the cells were further incubated at33°C following the addition of maintenance medium. At about 36 h later theinfected culture medium was harvested.PEG used for concentration. Using NT buffer, 50% (wIv) polyethylene glycol6,000 (Nakarai Chemical Products, Osaka) solution was prepared.Titration of virus infectivity. The plaque method described by Kisch (18)was employed with some modifications. The confluent HES cells in 60 mm plas-tic petri dishes were inoculated with 0.2 ml of virus appropriately diluted inEagle's MEM containing 0.5% gelatin. After 2 h at 33°C, cultures received 5 mlof agar overlay medium, which consisted of Eagle's MEM containing 5% FCS and0.8% agar, and were then incubated at 33°C. After 4 days at 33°C, an equalamount of identical agar overlay medium was added. Finally 5 ml of overlaymedium containing 0.01 % neutral red were added on the 8th day after inoculationand the plaques were counted on the following day.Protein determinations. With bovine serum albumin as the standard proteinmeasurements were made by the method of Lowry et al. (19).2Acta Medica Okayama, Vol. 32 [1978], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss4/2Purification of Respiratory Syncytial Virus 267RESULTSEffects of the sucrose concentration on the stabilization of RS virus. The inac-tivation curve was studied at 4°C in the presence of 10-40% sucrose (Fig. 1).00'10-l- 11II:JL.-"::;......0c0- 2Utt1L.-......0'1C:~L.-:J(/)-30 12 24 36 48HoursFig. 1. Effect of concentration of sucroseon RS virus infectivity at 4°e.7400/e20% 13300f015%E::Jl.L0.. 500'10-l43L.-_....L-_~_----'-_--Jo 12 24 36 48Hours after infectionFig. 2. Growth curve of RSvirus in HES cell culture.The half-reduction time of the infectivity titer was 7.7 h in NT buffer only, 17 hat a sucrose concentration of 10 %, but it was 24 to 26 h at a sucrose concentra-tion of 15% or over. Moreover, the inactivatio~ rate at the 12 h incubationtime was 76.1 % in NT buffer alone, 30.4 % in 10% sucrose-NT buffer, but abovea concentration of 15% sucrose-NT buffer it was less than 7.6% in every case.Thus as long as the entire procedure of concentration and purification is con-ducted within 12 h at 4°C, with a sucrose concentration of over 15%, the virusremains stable.Multiplication of RS virus in roller bottles. The release of infectious progenystarted 6 to 12 h after inoculation, increased exponentially up to 36 h, andreached its peak at the 48th h (Fig. 2). By 36 h a cytopathic effect, mainly infused cells, could be observed in the complete cell sheet. By 48 h a considerablenumber of cells became detached from the glass wall. Therefore, the culturemedium at the 36th h of cultivation is most suitable as substrate for concentrationand purification.3Ueba: Respiratory syncytial virus. I. Concentration and purification ofProduced by The Berkeley Electronic Press, 1978268 O. UEBAThe concentration by PEG. The culture fluid was immediately centrifuged at5,000 x g for 10 min to remove large cell debris (Fig. 3). The following pro-cedures were all conducted at 4°C or on ice. To the supernatant 50% PEG 6,000Infective tissue culture fluidIcentrifuged at 5,000 X g for 20 minIISediment(discarded)ISupernateIadd PEG 6,000 in NT buffer (final 10%) ;stir at 4°C for 60 min;centrifuged at 5,000 X g for 10 minIISedimentIsuspended in 20% sucrose-NT buffer;laid on 30% sucrose-NT buffer;centrifuged at 51,000 X g for 60 minIISupernate(discarded)ISupernate(discarded)ISedimentIsuspended in 20% sucrose-NT buffer;laid on 35, 45 and 60% sucrose density gradient in NT buffer;centrifuged at 165,000 X g for 60 minIcollect virus band on 45% sucrose-NT buffer;add NT buffer;laid on 30-60% sucrose density ~;radient in NT buffer;centrifuged at 165,000 X g for 180 minIcollect virus bandFig. 3. Schematic representation of the procedure used for the purification ofRS virus growth in HES cell cultures.TABLE 1. PURIFICATION OF RS VIRUSVol. PFU/ml Amt of PFU/mg RecoverySumple Protein of Protein(ml) (106) (mg/ml) (106) (%)Infective tissue culture fluid 1,200 5.5 2.40 2.2 100Cocentration with PEG 50 121 3. 74 32.4 92\_ .. _JResuspended pellet after high- ....-. ..,speed centrifugation 6,300 1. 29 4,900 95Band after centrifugation indiscontinuous sucrose density 0.5 4,400 0.77 5, 700 33gradientBand after centrifugation in linear O. 75 1, 700 0.25 6,800 19sucrose density gradient----_.__ ...._~.-4Acta Medica Okayama, Vol. 32 [1978], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss4/2Purification of Respiratory Syncytial Virus 269was added to make the final concentration 10%, and stirred for 60 min. Thenit was centrifuged at 5,000 x g for 10 min, and the sediment was suspended in20% sucrose-NT buffer with a dounce-homogenizer in 1/24 of the original mate-rial. Infectious virus collected amounted to 92 % of the original material (Table1).Purification by ultracentrifugation. The material concentrated with PEG 6,000was centrifuged through 30% sucrose-NT buffer (Fig. 3). There was hardlyany loss of infectious virus at this step of the purification (Table 1). Virus sus-pension was layered onto 35, 45 and 60% sucrose-NT buffer gradient, thencentrifuged at 165,000 x g for 60 min. A distinct band was observed on theboundary surface between 35% and 45% sucrose-NT buffer. This virus bandwas collected, layered on to a linear 30-60% sucrose·· NT buffer gradient, thencentrifuged at 165,000 x g for 180 min. One distinct band when formed slightlybelow the middle was collected from the bottom of the tube by fractionation.The infective virus was located in the region of density 1.18-1.20 g/cm3, and itspeak was at 1.19 g/cm3 (Fig. 4). Purified virus was obtained by pooling fractionsNos. 12,13 and 14. The infective titer of purified virus was 1.7 x 109 PFU and71.306 1.25(¥')1.20 E1.15 0'15E 1.10::::J 4I.LCl.co0 3X25 10 15 20Fraction numberFig. 4. Sucrose gradient analysis of RS virus.5Ueba: Respiratory syncytial virus. I. Concentration and purification ofProduced by The Berkeley Electronic Press, 1978~70 6. UEBAits recovery rate was 19%, while its protein concentration was 0.25 mg/ml.The ratio of infectivity to protein content was 6,800 x 106 PFU/mg in the puri-fied virus in contrast to 2.2 X 106 PFU/mg in the original material, showingan increase of about 3,aaO-fold.DISCUSSIONIn the concentration method of RS virus by Kanarek and Tribe favorableresults were obtained by using PEG 6,000 as with other paramyxoviruses (20).This method not only enables one to concentrate virus within a short period oftime, but also eliminate a large amount of the protein in the culture medium, soit is a very useful first step in purification. In the present experiment after thecompletion of this step the specific infectivity rose l5-fold.No attempt was made to purify infectious RS virus until the experiments ofWunner and Pringle (11). They used the method of resuspending the pellet con-centrated with PEG 6,000 in NTE buffer or HEPES buffer, and immediatelycentrifuging by isopycnic banding in sucrose or metrizamide. However theydescribed only a partial purification, and while the recovery rate at each stepremains unclear, resuspension in NTE buffer and HEPES buffer as well as a longtime-centrifugation must have resulted in considerable viral inactivation. Le-vine's purification (12) of RS virus using Hank's balanced salt solution (HBSS)demonstrated that the infectivity of RS virus did not diminish by treating it at4°C for 18 h. However, Hambling (13) found that the infectivity of RS virusfell to 1/10 in HBSS after 48 h, and we also observed a considerable reduction ininfectivity after 12 h in HBSS, hence we consider HBSS to be a poor stabilizer.The purification procedure employed by Levine requires about 60-69 handduring that time a considerable inactivation must have occurred.For purification without loss of infectivity, it is important that a mediumsuitable for purification be selected from among the stabilizers of RS virus.Inorganic salts such as MgS04 and NaCl at high concentration (14,15), areknown to be stabilizers of RS virus, but are not so suitable since they induceagglutination of intracellular structural conponents and inhibit fractionation. Onthe other hand, according to Law and Hull (16), a virus stock preserved for along period of time at - ;O°Cin 44.5% sucrose solution was very stable and evenat 4°C it was stable for up to 24 days. Tai et al. (17) also state that the infectivetiter is best at a preservation temperature of -60°C in 45% sucrose, and thatthe infective titer at 4°C does not decrease for 7 days. Sucrose, which seems tobe a suitable stabilizer of RS virus as mentioned above, has been widely employedfor the purification and fractionation of viruses and cellular components duringultracentrifugation. With this in mind the author studied the use of sucroseduring the purification of RS virus. Since the minimal time required for the6Acta Medica Okayama, Vol. 32 [1978], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss4/2Purification of RespIratory Syncytial VIrus 271purification of virus is 12 h, the minimal sucrose concentration which would notdiminish the infectivity was studied and it proved to be over 15%. Therefore,20 % sucrose-NT buffer was used as the suspension solution and in all other stepsover 20% sucrose-NT buffer was used.In the step where the virus concentrated with PEG 6,000 was centrifugedthrough 30% sucrose-NT buffer, there was no decrease in the infectivity, andit was possible to concentrate to 50-fold. Moreover, when a considerable amountof cellular components remained on 30 % sucrose-NT buffer the specific infectiv-ity rose 151-fold, indicating that it is an excellent step for concentration andpurification. To remove as much cellular components as possible the purificationwas accomplished by discontinuous sucrose density gradient, before the final stepof purification with a linear sucrose density gradient. At this step there was afaint band on 35% and 60% sucrose-NT buffer, and it was possible to removesome cellular components from the virus band at 45% sucrose-NT buffer stage.The protein content was reduced about 60 % in the earlier steps. The recoveryrate of infectious virus is high and the infectivity titer increased in the final stepwith a linear sucrose density gradient. Accordingly, the method described hereproved to be effective for the concentration-purification of infectious RS virus.For the elucidation of the relationship of virus structural component to theirbiological activity in the future large amounts of purified virus will be required.The present purification procedure suits this requirement.Acknowledgment. The author is indebted Prof. j. Tawara, Dr. H. Kumon and Dr. K.Akatsuka for providing many helpful suggestions.REFERENCES1. Chanock, R. M., Kim, H. W., Vargosko, A. j., Deleva, A., johnson, K. M., Cumming, C.and Parrott, R. H.: Respiratory syncytial virus. 1. Virus recovery and other observationsduring 1960 outbreak of bronchiolitis, pneumonia, and minor respiratory diseases in chil-dren. ]. Am. !vIed. Assoc. 176, 647-653, 1961.2. Waterson, A. P. and Hobson, D.: Relationship between respiratory syncytial virus andnewcastle disease-parainfluenza group. Br. med.]. 11, 1166-1167, 1962.3. Hodes, D. S., Schauf, V. and Chanock, R. M.: Isolation of RNA species from HeLa cellsinfected with respiratory syncytial virus. Proc. Soc. Exp. BioI. Med. 146, 287-290, 1974.4. Bussell, R. H., Waters, D. j. and Seals, M. K.: Measles, canine distemper and respiratorysyncytial virions and nucleocapsids. A comparative study of their structure, polypeptideand nucleic acid composition. Med. Microbiol. Immunol. 160, 105-124, 1974.5. jawetz, E., Melnick, j. L. and Abelberg, E. A.: Review of Medical Microbiology. LangeMedical Publication, Los Altos, California, pp.29l-295, 1976.6. joncas, j., Berthiaume, L. and Pavilanis, V.: The structure of the respiratory syncytialvirus. Virology 38, 493-496, 1969.7. Norrby, E., Marusyk, H. and Orvell, C.: Morphogenesis of respiratory syncytial virus ina green monkey cell line (Vero). ]. Virol. 6, 237-242, 1970.8. Bachi, T. and Howe, C.: Morphogenesis and ultrastructure of respiratory syncytial virus.]. Virol. 12, 1173-1180, 1973.7Ueba: Respiratory syncytial virus. I. Concentration and purification ofProduced by The Berkeley Electronic Press, 1978272 O. UEBA9. Richman, A. V., Pedreira, F. A. and Tauraso, N. M.: Attempts to demonstrate hemag-glutination and hemadsorption by respiratory syncytial virus. Appl. Microbiol. 21, 1099-1100, 1971.10. Chanock, R. M., Roizman, B. and Myers, R.: Recovery from infants with respiratoryillness of a virus related to chimpanzee coryza agent (CCA). L Isolation, properties andcharacterization. Am.]. Hyg. 66, 281-290, 1967.11. Wunner, W. H. and Pringle, C. R.: Respiratory syncytial virus proteins. Virology, 73, 228-243, 1976.12. Levine, S.: Polypeptides of respiratory syncytial virus. J. Virol. 21, 427-431, 1977.13. Hamgling, M. H.: Survival of the respiratory syncytial virus during storage under vari-ous conditions. Br.]. Exp. Pathol. 45, 647-667, 1964.14. Yamamoto, K.: Stabilizing effect of inorganic salts on RS virus. ]pn.]. Med. Sci. Bioi.22, 337-339, 1969.15. Rechsteiner, J.: Thermal inactivation of respiratory syncytial virus in water and hyper-tonic solutions. ]. gen. Viro!. 5, 397-403, 1969.16. Law, T. J. and Hull, R. N.: The stabilizing effect of sucrose upon respiratory syncytialvirus infectivity. Proc. Sac. Exp. Bioi. Med. 128, 515-518, 1968.17. Tai, F. H., Chu, C. Z., Chi, W. H. and Chu, S.: Studies of respiratory syncytial virus. 2.Stabilization of respiratory syncytial virus. Chin.]. Microbiol. 7, 20-24, 1974.18. Kisch, A. L. and johnson, K. M.: A plaque assay for respiratory syncytial virus. Proc.Soc. Exp. BioI. Med. 112, 583-588, 1963.19. Lowry, D. H., Rosebrough, N. j .. Farr, A. L. and Randall, R. j.: Protein measurementwith the Folin phenol reagent. ]. BioI. Chern. 193, 238-275, 1951.20. Kanarek, A. D. and Tribe, G. W.: Concentration of certain myxoviruses with polyethyleneglycol. Nature 214, 927-928, 1967.8Acta Medica Okayama, Vol. 32 [1978], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss4/2

Respiratory syncytial virus. I. Concentration and purification of the infectious virus

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