Henry Ford Hospital Medical Journal

Volume 10 | Number 3 Article 9

9-1962

Inactivation Of Vacuolating Virus (SV 40) By Beta-propiolactone I. Evaluation In Tissue CultureHajime Hayashi

Gerald A. LoGrippo

Follow this and additional works at: https://scholarlycommons.henryford.com/hfhmedjournal

Part of the Life Sciences Commons, Medical Specialties Commons, and the Public HealthCommons

This Article is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in HenryFord Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons. For more information, please contactacabrer4@hfhs.org.

Recommended CitationHayashi, Hajime and LoGrippo, Gerald A. (1962) "Inactivation Of Vacuolating Virus (SV 40) By Beta-propiolactone I. Evaluation InTissue Culture," Henry Ford Hospital Medical Bulletin : Vol. 10 : No. 3 , 463-470.Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol10/iss3/9

Henry Ford Hosp. Med. Bull. Vol. 10, September, 1962

INACTIVATION OF VACUOLATING VIRUS (SV 40) BY BETA-PROPIOLACTONE

I. EVALUATION IN TISSUE CULTURE

H A J I M E HAYASHI, Ph.D. AND GERALD A. LOGRIPPO, M.D.

SIMIAN VIRUS 40 (SV 40) has been of great concern to virologists and clinicians since Sweet et al.'" reported in 1960 the vacuolating virus from cell cultures of rhesus and of cynomolgus monkey kidney. It was shown as a viable contaminant of poliomyelitis and adenovirus vaccines, cultured from rhesus and cynomolgus kidney cells."'" SV 40 has been called "vacuolating virus" by Sweet et al." because of its prominent cytoplasmic vacuolation, seen in infected kidney cultures of Cercopethecus aethiops (African grivet monkey) and is considered unique among simian viruses. In addition, it was reported that SV 40 showed cytopathic effects (CPE) on primary cultures of several human tissues."

It is of growing interest among virologists that SV 40 is oncogenic as shown by Eddy and associates.' They reported the appearance of undifferentiated sarcomas in subcutaneous tissues, kidneys and lungs of hamsters which had been inoculated within three days after birth with the cell extracts of frozen and ground primary cell cultures of rhesus monkey kidney. Such tumors appeared within six to nine months after inoculation. In subsequent publications^'^ Eddy and coworkers identified SV 40 in the kidney extract as responsible for tumor formation in hamsters. Further­more, Girardi et al.^ also showed that SV 40 induced single or multiple fibrosarcomas at the site of injection which were histologically of varying degrees of malignancy in hamsters inoculated at birth. Other oncogenic viruses are now recognized and Melnick'^ has proposed the name "Pa-po-va" for the oncogenic group which includes the papilloma virus (in rabbit and human warts), polyoma virus and vacuolating virus (SV 40). This new group is classified on their similarities in virus properties such as size, stability, DNA nature, etc. as well as their oncogenicity.

Although these findings should be of concern to the clinician, they should not be alarming, as no clinical signs and symptoms in man have been reported from SV 40 exposure. Morris et al." mentioned that none of 35 adult volunteers showed any clinical manifestations after SV 40 was administered by the respiratory route.

•Department of Laboratories

463

HAYASHI AND LOGRIPPO

SV 40 was recovered from throat specimens in three of the volunteers on seven to eleven days after exposure. In addition virus neutralizing antibody against this agent appeared in the serum in 22 of 35 volunteers. Melnick and Stinebaugh'" also reported experiments on 3- to 6-month-old infants who were fed SV 40 found in the polio­myelitis oral vaccine. They showed that some of the infants excreted vacuolating virus for four weeks and that the vacuolating virus seemed to interfere in some degree with the multiphcation of the poliomyelitis virus in the gastrointestinal tract, as SV 40 carriers excreted less polio virus for shorter intervals of time than did those infants who did not become infected by the SV 40.

Since the SV 40 is more resistant to formalin treatment than is the poliomyelitis and adenoviruses in vaccine production,"'" new methods for SV 40 inactivation are being sought. This is essential since SV 40 is considered a potential hazard. The purpose of this presentation is to appraise the usefulness of beta-propiolactone (BPL) in the inactivation of SV 40 and the value of this drug in vaccine production problems. The usefulness of BPL in cold sterilization of biological materials' '"'" '̂ and in the preparadon of inactivated virus vaccines"''"'" has been reported.

MATERIALS AND METHODS

Cell Cultures

A continuous kidney cell line of Cercopithecus aethiops, African green monkey (BS-C-1)' was obtained from Mrs. H. E. Hopps, Division of Biologies Standards, National Institutes of Health, Bethesda, Md. Growth medium for these cells consists of M199,'5 0.1 per cent yeastolate (Difco), 20 per cent fetal bovine serum and antibiotics (100 tig of streptomycin and 100 units of penicillin per ml). Maintenance medium consists of MI99 with the same concentradon of antibiotics. Tube and bottle cultures were prepared by the trypsinization procedure.

Virus

Simian virus 40 (SV 40) strain A 426-RN-2 supplied by Dr. B. E. Eddy, Division of Biologies Standards, NIH, was serially propagated on BS-C-I cells in this laboratory. The TCID50 of this agent in suspension was at least lO"'-̂ per ml and all experiments showed this virus titer or higher. In addition, virus infected cells were employed to test the concentration of BPL necessary to penetrate and inactivate SV 40 intracellularly. For this study 100,000 cells per ml were treated while suspended in the original undiluted tissue cultured virus suspension. This preparation was harvested at the peak phase of CPE.

Beta-propiolactone* (BPL) solutions

From a 10 per cent I(wt/voI) (Sp. Gr. BPL-1.149)] BPL solution prepared in cold distilled water, all serial dilutions were made immediately before addition to virus suspension or virus infected cell suspensions in proportional amounts necessary for final concentrations. Since BPL hydrolyzes rapidly in water, all solutions must be freshly prepared in cold distilled water in order to maintain the maximum amount of the lactone form of the drug.'

Conditions for virus inactivation

The principles employed for treating humaii plasma against the hepatitis virus'''" were used in these studies. Undiluted virus-drug mixtures were made at 4-9°C. by keeping all materials in ice baths. All mixtures were intermittendy and vigorously shaken for 3-5 minutes, then allowed to stand at 4-9°C. for at least one hour. Such mixtures were then incubated at 37°C. for two hours for rapid hydrolysis and action of BPL Infected cell-drug mixtures were similarly treated. Since BPL forms acid products on hydrolysis,

*Betaprone: "Specially purified" brand of beta-propiolactone, available only from Testagar & Co. Detroit, Michigan. ''

464

SV 40 INACTIVATION BY BPL

sterile 1 N. NaOH solution was used for pH adjustments (7.0-7.2) in proportion to the rate of acids formed. This was readily accomplished by ths phenal red indicator systsm prssent in tissue culture fluid.

SV 40 infectivity tests

To each BS-C-1 tube culture, 0.2 m.l. of virus-drug or infected cell-drug mixture was added and incubated at 37°C. in stationary position or on roller drums. Tissue culture fluid from tubes with nsgative CPE was subcultured twics when indicated. In bulk tssts. 10 ml. (20 per cent of volume treated) of virus-drug or cell-drug mixtures wsre added to BS-C-1 csUs grown in 32 oz. prsscription botdes. Although CPE for SV 40 was observed betwesn 3 to 8 days, all test materials were maintained for 14 to 28 days, dependsnt upon ths degsneration tims for the normal cell cultures in the respective tests. Parallel controls with normal cell cultures were conducted with equivalent amounts of the degeneration products formed from 0.35 per cent BPL in maintenance medium. Such quantities were not deletsrious to the cell cultures.

RESULTS

The efficacy of BPL in inactivating the SV 40 in vitro is evaluated by its ability to inhibit the CPE on BS-C-1 cells (Figure l a ) . The cells become large.

'Mr

Figure la Arrows indicate areas of characteristic CPE from SV 40 infection on BS-C-1 cell cultures (X450)

rounded or elongated and swollen with multiple vacuoles. Disruption of the normal cell monolayer with detachment of cells from the glass surface is readily observed in Figures la and lb. This effect becomes apparent in 3-8 days. Figure lb is an enlarged area of SV 40 infected cells showing the vacuolation in more detail. Normal cells are shown in Figure 2. They appear as a monolayer sheet of cells in pavement­like arrangement with no detachment from the glass surface.

465

HAYASHI AND LOGRIPPO

«

* Figure lb

Enlargement of areas showing cellular vacuoles indicated by arrows in Figure la (X1935)

If

s

Figure 2 Normal monolayer sheet of BS-C-1 cell cultures {X450)

466

SV 40 INACTIVATION BY BPL

Since 17 animal viruses have been inactivated with 0.4 per cent or less concen­tration of BPL", 0.35 per cent and less was selected for range of inactivation for the SV 40. Table I shows the preliminary results of varying concentrations of BPL from 0.35 per cent to 0.125 per cent. The table also shows the concentration of BPL required to inactivate the virus suspension in undiluted tissue culture fluid. In addition, the quantity of BPL is shown which is required to penetrate monkey

Table I Effect of Beta-propiolactone (BPL) on Simian Virus 40 (SV 40)

Percent

BPL concentrations

Cytopathic Effects on BS-C-1 Cells Percent

BPL concentrations SV 40 in TC f lu id SV 40 infected cell

Percent

BPL concentrations suspension suspension

0.35 0/4* 0/4

0.20 0/4 2/4

0.10 4/4 4/4

0.05 4/4 4/4

0.025 4/4 4/4

0.0125 4/4 4/4

*Numerator indicates numbers of tubes with positive CPE and denominator numbers of tubes tested.

kidney cells laden with intracellular virus suspended in its originally infected tissue culture fluid. In columns 1 and 2 of Table I , the inactivation concentration lies between values 0.2 per cent and 0.1 per cent BPL when the virus is in a cell free tissue culture fluid. However, BPL concentrations between 0.35 per cent and 0.2 per cent are required to penetrate infected cells and achieve the same degree of virus inactivation.

The data seen in Table I I are the results of two subsequent experiments which indicate that 0.14 per cent BPL is sufficient to inactivate the virus suspension but that 0.35 per cent of BPL is the minimum concentration necessary to penetrate the cells and inactivate SV 40.

Table I I Effect of Beta-propiolactone on SV 40 (Data from two experiments: tube method)

Percent BPL concentrations

Cytopathic Effects on BS-C-1 cells Percent

BPL concentrations SV 40 in TC f lu id SV 40 infected cell

Percent BPL concentrations

suspension suspension

0.35 N T * 0/8**

0.30 N T 5/8

0.25 N T 8/8

0.20 N T 8/8

0.18 0/8** N T

0.16 0/8** N T

0.14 0/8** N T

0.12 8/8 N T

0.10 8/8 N T

*NT: Not tested. **Indicates negative CPE in three successive cultures.

467

HAYASHI AND LOGRIPPO

Table I I I is a summary of three experiments in which larger volumes of virus-drug mixtures of infected materials were tested in larger containers with greater numbers of host cells. Such bulk tests serve to demonstrate trace quantities of virus which can be manifested only by testing larger samples of infected materials. In

Table I I I Effect of Beta-propiolactone on SV 40 (Data from three experiments: bulk test)

Percent BPL concentrations

Cytopathic Effects on BS-C-1 cells Percent

BPL concentrations SV 40 in TC fluid Suspension

SV 40 infected cell Suspension

0.35 NT* 0/4

0.20 0/3** NT 0.18 0/3 NT 0.16 0/3 NT

0.14 1/3 NT

*NT: Not tested. **Numerator indicates numbers of bottles with positive CPE, and denominator numbers of bottles

tested.

the undiluted tissue culture fluid, results indicate that 0.16 per cent BPL concen­tration is necessary for complete inactivation of SV 40 whereas in the tube method 0.14 per cent was effective; indicating the presence of trace virus quantities not detectable in the tube test. However, in the infected cell suspension, the effective concentration of BPL remained 0.35 per cent as indicated in the tube test.

DISCUSSION

One of the reasons for this study was to ascertain whether the concentration of BPL necessary to inactivate the SV 40 would be less than that necessary to inactivate the poliomyelitis and adenoviruses in tissue culture suspensions. In previous publication" it was shown that 0.3 per cent BPL inactivated the poliomyelitis virus by the tube method but that 0.4 per cent was required to eliminate trace quantities as shown in the bulk method. The inactivating concentration for adenovirus (Type 3) was 0.3 per cent by the bulk method. The SV 40 is more susceptible to BPL concentration than either poliomyelitis or adenoviruses, this is the converse to that found with formalin method for virus vaccine preparations."'" As has been reported from this laboratory, BPL has greater virucidal potency than formalin" and BPL combined with ultraviolet irradiation has synergistic virucidal effectivities' with anti­genic potency equivalent to live vaccines compared in parallel tests. Therefore, the use of BPL is a logical suggestion to the SV 40 problem in virus vaccine production.

In addition, the results showed the penetrability of BPL into infected cells with inactivation of SV 40.

Of further interest is the fact that SV 40 is an oncogenic agent. These results suggest a wider field of usefulness for sterilization with BPL. Merely evaluating

468

SV 40 INACTIVATION BY BPL

the inactivation of SV 40 in tissue culture is not conclusive evidence that the oncogenic

properties have been destroyed. Therefore, such inactivated material must be evaluated

in suckling hamsters. These results are forthcoming in subsequent publications.

SUMMARY

1. Beta-propiolactone was found to inactivate the simian virus 40 (SV 40) in

tissue culture fluid and infected cell suspensions at the concentrations of

0. 16 per cent and 0.35 per cent (wt/vol) respectively.

2. These tests were evaluated in continuous kidney cell cultures of Cercopethecus

aethiops (BS-C-1).

3. The present status of the SV 40 problem in virus vaccine production was

reviewed, and the value of BPL in these problems was discussed.

REFERENCES

1. Eddy, B. C, Borman, G. S., Berkeley, W. H., and Young, R. D.: Tumor induced in hamsters by injection of rhesus monkey cell extracts, Proc. Soc. Exper. Biol. & Med. 107:191, 1961.

2. Eddy, B. E.: Tumors produced in hamsters by SV 40. Fed. Meetings, Symposium on virus and neoplasia, April 1962.

3. Eddy, B. E., Borman, G. S., Grubbs, G. E., and Young, R. D.: Identification of the oncogenic substance in rhesus monkey kidney cell cultures as simian virus 40, Virology 17:65, 1962.

4. Gerber, P., Hottle, G. A., and Grubbs, R. E.: Inactivation of vacuolating virus (SV 40) by formaldehyde, Proc. Soc. Exper. Biol. & Med. 108:205, 1961.

5. Girardi, A. N., Sweet, B. H., Slotnick, V. B., and Hilleman, M. R.: Development of tumors in hamsters inoculated in the neonatal period with vacuolating virus, SV 40, Proc. Soc. Exper. Biol. & Med. 109:649, 1962.

6. Hartman, F. W., and LoGrippo, G. A.: Combined beta-propiolactone and ultraviolet irradiation for plasma sterilization. In Henry Ford Hosp. Symposium: Hepatitis Frontiers, Ch. 33. Little, Brown & Co., Boston, 1957.

7. Hopps, H. E., Bernheim, B. C, Nisalak, A., and Smadel, J. E.: Biological characteristics of a serially cultivated kidney cell culture derived from the African green monkey, Cerco­pithecus aethiopics. Fed. Proc. 21:454, 1962.

8. LoGrippo, G. A., and Hartman, F. W.: Antigenicity of beta-propiolactone-inactivated virus vaccines, J. Immunol. 75:123, 1955.

9. LoGrippo, G. A.: Antigenicity of combined beta-propiolactone and ultraviolet inactivated virus vaccines, J. Immunol. 80:198, 1957.

10. LoGrippo, G. A., and Rupe, C. E.: Chemical sterilization of whole blood and plasma: beta-propiolactone. In Henry Ford Hosp. Symposium: Hepatitis Frontiers, Ch. 31. Little, Brown & Co., Boston, 1957.

11. LoGrippo, G. A.: Investigations of the use of beta-propiolactone in virus inactivation, Ann. N. Y. Acad. Sci. 83:578, 1960.

12. LoGrippo, G. A.: The safety of beta-propiolactone as a biologic sterilizing agent: clinical evaluation with human plasma and homotransplants, Angiology 12:80, 1961.

13. Melnick, J. L.: Papova virus group. Science 135:1128, 1962.

14. Melnick, J. L., and Stinebaugh, S.: Excretion of vacuolating SV-40 virus (Papova virus group) after ingestion as a contaminant of oral poliovaccine, Proc. Soc. Exper. Biol. & Med. 109:965, 1962.

15. Morgan, J. F., Morton, H. J., and Parker, R. C : Nutrition of animal cells in tissue culture. 1. Initial studies on a synthetic medium, Proc. Soc. Exper. Biol. & Med. 73:1, 1950.

469

HAYASHI AND LOGRIPPO

16. Morris, J. A., Johnson, K. A., Aulisio, C. G., Chanock, R. M., and Knight, V.: Clinical and serologic responses in volunteers given vacuolating virus (SV 40) by respiratory route, Proc. Soc. Exper. Biol. & Med. 108:56, 1961.

17. Peck, F. B., Jr., Powell, M. H., and Culbertson, C. G.: Duck embryo rabies vaccine; study of fixed virus vaccine grown in embryonated duck eggs and killed with beta-propiolactone (BPL), J.A.M.A. 162:1373, 1956.

18. Powell, H. M., and Culbertson, C. G.: Antirabies vaccine prepared from fixed virus grown in embryonated duck eggs and killed with beta-propiolactone, Bacteriol. Proc. pp 70, 1956.

19. Shein, H. M., and Enders, J. F.: Multiplication and cytopathogenicity of simian vacuolating virus 40 in cultures of human tissues, Proc. Soc. Exper. Biol. & Med. 109:495, 1962.

20. Sweet, B. H., and Hilleman, M. R.: The vacuolating virus, SV 40, Proc. Soc. Exper. Biol. & Med. 105:420, 1960.

470