APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1981, p. 718-723 Vol. 41, No. 30099-2240/81/030718-06$02.00/0

Mechanism of Enteroviral Inactivation by OzoneD. ROY,lt* P. K. Y. WONG,2 R. S. ENGELBRECHT,' AND E. S. K. CHIAN3

Departments of Civil Engineering' and Microbiology,2 University of Illinois, Urbana, Illinois 61801, andGeorgia Institute of Technology, Atlanta, Georgia 303323

The mechanism of enteroviral inactivation by ozone was investigated withpoliovirus 1 (Mahoney) as the model virus. Ozone was observed to alter two ofthe four polypeptide chains present in the viral protein coat of poliovirus 1.However, the alteration of the protein coat did not significantly impair virusadsorption or alter the integrity of the virus particle. Damage to the viral RNAafter exposure to ozone was demonstrated by velocity sedimentation analysis. Itwas concluded that the damage to the viral nucleic acid is the major cause ofpoliovirus 1 inactivation by ozone.

Ozone has been known for years to be a strongdisinfecting agent. Although the ability of ozoneto inactivate enteric viruses has been docu-mented in the literature by many researchers,little attention has been directed toward thedetermination of the mechanism of ozone inac-tivation of viruses. Information regarding themechanism of inactivation would be useful indeveloping mathematical models to predict theperformance of the ozone disinfection process asapplied to the inactivation of viruses in waterand wastewater treatment.

Enteroviruses consist of a single-stranded ri-bonucleic acid (RNA) of 2 x 106 daltons enclosedin a protein coat consisting of 60 capsomeres.The protein coat is about 6 nm thick with a totalmolecular weight of 5.7 x 106. Each capsomerehas a molecular weight of approx2imately 95,000and is made up offour polypeptide chains havingmolecular weights of 35,000, 28,000, 24,000, and8,000 (7).

Poliovirus infection is initiated by adsorptionof virus onto the host cells. After the uncoatingof the protein coat, viral RNA is released intothe cell cytoplasm to initiate the synthesis of theproteins and the replication of RNA. After aneclipse phase, as reported by Martin and Work(11), viral RNA, protein, and then the matureviruses appear. It is evident that any compound,to be effective as a virucidal agent, should becapable of causing physical disruption of thevirion or reacting with the protein coat or theviral RNA or both such that one or more of thesteps of viral replication is blocked.The objective of this research was to elucidate

the mechanism of enteroviral inactivation byozone by identifying the major reaction(s) andthe lethal site(s) involved in the inactivationprocess.

t Present address: Department of Civil Engineering, Louis-iana State University, Baton Rouge, LA 70803.

MATERIALS AND METHODSGlassware. All glassware was thoroughly cleaned

to ensure that it was ozone demand free. It was cleanedwith a dichromate-sulfuric acid mixture, rinsed in tapwater followed by distilled water, and finally soakedin a strong ozone solution before drying at 180°C forat least 6 h to ensure the elimination of ozone.

Virus. Poliovirus type 1 (Mahoney), originally ob-tained from Gerald Berg, Environmental ProtectionAgency, Cincinnati, Ohio, was used as the model en-teric virus.

Cell line. An African green (Cerocopithecus ae-thiops) monkey kidney cell line known as BGM (buf-falo green monkey) was used for virus propagation andtitration by plaque assay. BGM cells were grown inmedium 199 (Grand Island Biological Co., Grand Is-land, N.Y.) supplemented with 10% fetal calf serum(Grand Island Biological Co.), 0.2% sodium bicarbon-ate, and 1% antimycotic antibiotic (Grand Island Bio-logical Co.). The cell line was maintained by continu-ous passage at a concentration of 1.2 x 106 cells perml.Ozone demand-free water. Distilled deionized

water was ozonated for 15 min to oxidize any traceorganic matter and then boiled for 30 min to dissipateresidual ozone. The treated water was then storedunder ultraviolet light until used.

Buffers. Buffers to achieve the two pH values usedin this study were prepared as follows: (i) (pH 7.2)1.967 g of sodium dihydrogen phosphate (NaH2PO4,H20) and 5.074 g of anhydrous disodium hydrogenphosphate (Na2HOP4) were dissolved in 5 liters ofozone demand-free water; (ii) (pH 4.3) 2.6 ml of con-centrated acetic acid (CH3COOH) and 0.645 g of an-hydrous sodium acetate (CH3COONa) were dissolvedin 5 liters of ozone demand-free water.

Phosphate-buffered saline solution. Concen-trated (10 times) phosphate-buffered saline solutionwas prepared by the procedure described by Rovozzoand Burke (15).

Preparation and purification of viruses. Flaskscontaining a confluent monolayer of BGM cells werewashed twice with serum-free growth medium andinoculated with a stock virus suspension. Virus ad-sorption was allowed to occur for 1 h in an atmosphereof 5% CO2 at 37°C. After 24 to 36 h of incubation and

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ENTEROVIRAL INACTIVATION BY OZONE 719

before further purification, each flask was frozen at-70°C.The crude suspension containing virus-infected cells

was purified by a technique similar to that describedby Scarpino et al. (18). In this procedure, the crudesuspension was frozen at -70°C and thawed threetimes, followed by centrifugation at 10,000 rpm for 1h to remove the cell debris. The clarified supernatantwas frozen once again at -70°C, thawed, and thencentrifuged at 40,000 rpm for 5 h. The pelleted viruswas suspended in buffer appropriate for the intendedexperiments. The total organic carbon of such a viruspreparation, having a titer of 106 plaque-forming unitsper ml, was determined by an ultralow total organiccarbon analyzer (Dohrman, Envirotech, Santa Clara,Calif.) to be on the order of 270 ,ug/liter. This level oftotal organic carbon was found to exert negligibleozone demand in batch disinfection system as used inthis study (16).

Labeled virus was prepared by growth in mediumcontaining either '4C-amino acid mixture at 5 ,uCi/ml(Amersham Corp., Arlington Heights, Ill.) or [3H]uri-dine at 10 MCi/ml for 24 h and purified as describedabove.

Virus assay. Virus titers were determined byplaque assay, with confluent BGM monolayers in tis-sue culture plates (60 by 15 mm; Falcon Division,Bioquest, Oxnard, Calif.). Serial dilutions of the virussuspension were prepared with Hanks balanced saltsolution (Grand Island Biological Co.), containing 2%fetal calf serum, 0.5% thiosulfate, 1% antimycotic an-tibiotic, and 0.5% sodium bicarbonate. After inocula-tion and adsorption at 37°C under 5% CO2 for 1 to 2 h,the cells were overlaid with 5 ml of agar medium,consisting of medium 199 plus 2% fetal calf serum, 1%magnesium chloride, 1% antimycotic antibiotic, 0.5%sodium bicarbonate, and 0.9% agar, and reincubatedat 37°C until the appearance of plaques. At this time,the monolayer was fixed with 70% ethanol, formalde-hyde, and acetic acid in a volume ratio of 20:1:1. Themonolayer was then stained with a 1% solution ofcrystal violet, and plaques were counted.Gel electrophoresis. Electrophoretic analysis of

the poliovirus capsid polypeptides labeled with 14C-amino acids was performed by the sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresismethod described by Vrijsen and Boeye (20). Gelcolumns, 9 cm long, containing 12.5% polyacrylamide,0.1% SDS, and 0.1 M phosphate buffer, were preparedin glass tubes having an inside diameter of 6 mm. ThepH of the gel was 7.2. Each sample, consisting of 0.3ml, was layered on gel, and electrophoresis was per-formed with a conventional gel electrophoresis cell for24 h at 25 V with both electrode buffers at pH 7.2.

After electrophoresis, the gels were sliced using aMickle gel slicer (Brinkman, Des Plaines, Ill.), andslices were transferred into glass vials, solubilized in0.5 ml of hydrogen peroxide at 65°C for 24 h, andradioactivity was determined by scintillation spec-trometry.

Extraction and analysis of viral RNA. ViralRNA was extracted from purified poliovirus by themethod developed by Mandel (10), who showed thatthe efficiency of RNA extraction after disruption ofthe capsid by SDS is greatest at acidic pH valuesranging from 3.5 to 4.4. In view of this, extraction and

analysis of viral RNA was performed at pH 4.3. Aceticacid buffer at pH 4.3 (2.4 ml) and 10% SDS solution(0.3 ml) were added to 0.3 ml of viral suspension. Themixture was held at room temperature for 1 h andthen centrifuged on a 5 to 20% sucrose density gradientat 35,000 rpm for 210 min at 20°C.

Viral attachment experiment. Virus samples la-beled with either [3H]uridine or '4C-amino acids wereinoculated on BGM cell monolayers and incubated for60 to 90 min in an atmosphere of 5% CO2 at 37°C.After incubation, the cell monolayers were washedthree times with phosphate-buffered saline solution toremove unattached viruses. The cell layer was thenremoved with a rubber policeman and collected in acentrifuge tube. The cell suspension was pelleted bycentrdfugation at 3,000 rpm for 20 min. The cell pellet,which did not contain any unattached viruses, wasthen suspended, and the associated radioactivity wasdetermined.Ozonation procedure. The experiments carried

out for elucidation of the mechanism of enteroviralinactivation by ozone were performed in a batch sys-tem using glass tubes at room temperature. The pro-cedures of ozone generation and preparation ofaqueous solution of ozone have been reported earlier(16). An aqueous solution of ozone of known concen-tration was added to the tube containing radioactiveviruses and was manually mixed for a predeterminedcontact time. An appropriate volume of 0.5 M sodiumthiosulfate solution was then added to the tubes toneutralize any residual ozone. Samples were then an-alyzed as required for the particular study.

Survival of viruses after exposure to ozone is de-pendent on the residual ozone concentration. Kineticsof inactivation of poliovirus 1 using various residualozone concentrations have been presented earlier (16).It was observed that a 0.21-mg/liter residual ozoneconcentration at pH 7.2 for 30 s resulted in approxi-mately 99% inactivation of poliovirus 1. Similar studiesby Katzenelson et al. (6) reported that between 0.1 to0.2 mg/liter ozone residual, the percent survival de-creased with increasing residual concentration. How-ever, increasing the ozone residual from 0.2 to 1.5 mg/liter did not alter the percent survival. In view of theabove findings, residual ozone concentrations weremaintained in the range of 0.3 to 0.8 mg/liter in allexperiments of this study to insure at least 99% inac-tivation of viruses.The spectrophotometric method of ozone measure-

ment developed by Schechter (19) was used to monitorthe aqueous concentration of ozone.

RESULTSEffect of ozone on structural integrity of

the virus. To determine whether the overallintegrity or the structure of a virus particle wassignificantly altered after exposure to a low con-centration of ozone, a preparation of [3H]uri-dine-labeled poliovirus 1 was exposed to 0.32-mg/liter residual ozone concentration for 30 s atroom temperature. Untreated and ozone-treatedlabeled virus preparations were then centrifugedon 10 to 40% sucrose gradients for 2 h at 35,000rpm at 50C and then fractionated, and radioac-

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tivity was determined. As shown in Fig. 1, bothozone-treated and untreated virus preparationsyielded a single peak of radioactivity, indicatingan essentially homogeneous preparation of in-tact virus particles. The sedimentation coeffi-cient for the peak was calculated to be 158S bythe approximate method of Griffith (5); thisvalue agrees with the range of 153 to 160S re-ported for single poliovirus particles by Ruckert(17).

If [3H]uridine-labeled viruses were so severelydamaged by ozone as to cause disintegration,then the nucleic acid would have leaked out, inwhich case nucleic acids having a much lowermolecular weight would have sedimented as aband at a different location. These results sug-gest that the physical integrity of the virus didnot change significantly as a result of exposureto ozone, i.e., the majority of the viral populationremained as single intact virions.

Effect of ozone on viral protein. Althoughthere was no evidence that the viral protein coatwas dissociated under these conditions (Fig. 1),partial damage or some change in one or morepolypeptide chains could not be ruled out. Todetermine whether any change in the viral cap-

2501

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50

° 350C

I 250

150

50

4 8 12 16 20 24 28 32 36 40TOP BOTTOM

FRACTION NO.

FIG. 1. Centrifugation analysis of [3HJuridine-la-beled poliovirus 1 on 10 to 40%o sucrose density gra-dients for 2 h at 35,000 rpm and 5°C for (A) controlvirus suspension; and (B) viruses exposed to 0.32-mglliter residual ozone concentration for 30 s.

sid did occur due to ozonation, viruses labeledwith '4C-amino acid were exposed to 0.7-mg/liter residual ozone concentration for 60 s andthen analyzed by SDS-polyacrylamide gel elec-trophoresis. Labeled viruses, not exposed toozone, were analyzed in parallel for comparison.The electrophoretic profile of the unexposedviruses (Fig. 2A) revealed three large peaks cor-responding to polypeptide chains VP1, VP2, andVP3, and a small peak, VP4. The structuralpolypeptides of unexposed viruses identified inthis experiment are in agreement with thosereported by Maizel et al. (9). Comparison of thisprotein profile with that of ozone-exposed virus(Fig. 2B) suggests possible damage to poly-peptide chains VP1 and VP2. In addition, therewas the appearance of a new peak with fractionno. 19, suggesting the possible breakdown ofthe large-molecular-weight polypeptide chains.Polypeptide chain VP4 appears to be unaffectedby ozone treatment. In the case of enteroviruses,it has been shown that VP4 is responsible forvirus attachment (1, 3, 4, 8). The protein profilesshown here and the adsorption studies discussedbelow indicate that inactivation of enterovirusesby a low dosage of ozone cannot be attributed tothe damage of the viral capsid polypeptide af-fecting attachment or penetration or both.

Effect of ozone on viral attachment-pen-etration. Inactivation of poliovirus 1 by ozonewas found to be associated with a change in thepolypeptide structure of the protein coat. Al-though such a change did not cause disintegra-tion of the viral particles, the possibility of af-fecting the attachment of the virus particles onthe host cells could not be ruled out. Afterattachment, viruses penetrate the host cells bya process which has not been completely iden-tified to date. Because of this difficulty, in thecurrent study these two processes, attachmentand penetration, are treated as one process.The polypeptide mapping, as discussed in the

previous section, indicated that the polypeptidechain VP4 remained unchanged as a result ofozonation. In view of this observation, two setsof experiments were performed to determinewhether ozone-exposed poliovirus showing achange in VP1 and VP2 but no change in VP4was affected with respect to its ability to attachto host cells.

In the first set of experiments, different sam-ples of the same preparation of poliovirus 1labeled with [3H]uridine were exposed to variousresidual ozone concentrations for 30 s at pH 7.2and inoculated onto cell monolayers. After in-cubation for attachment, cell-associated radio-activity was measured; the results are presentedin Fig. 3. Percent penetration-attachment wascalculated as the ratio of cell-associated radio-

A ( 158 S)

-B

I

720 ROY ET AL.

ENTEROVIRAL INACTIVATION BY OZONE 721

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0-

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BOTTOM

FRACTION NO.FIG. 2. SDS-polyacrylamide gel electrophoresis

pattern ofpoliovirus 1 proteins from (A) control virussuspension and (B) poliovirus 1 exposed to 0.7-mg/liter residual ozone concentration for 60 s. Polio-viruses were labeled with "4C-amino acid mixture,and gel electrophoresis wasperformed by the methoddescribed in the text.

zLuJ2 75

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FIG. 3. Effect on poliovirus I penetration-attach-ment toBGMhost cell after exposure to various ozoneresidual concentrations for 30 s at pH 7.2. Viruseswere labeled with [3HJuridine.

activity imparted by the labeled virus to that ofthe labeled control virus suspension. The datapresented in Fig. 3 indicate that the reduction inpercent virus penetration-attachment was notmarkedly affected by the exposure of the virusesto ozone residual concentrations up to 0.8 mg/liter.The second set of experiments was perforned

with 0.51-mg/liter residual ozone concentrationat pH 7.2; '4C-amino acid-labeled poliovirus wasexposed to this residual concentration of ozonefor different contact times. The results of thisset of experiments are presented in Fig. 4. Thereduction in percent penetration-attachment ob-tained in this set of experiments was higher thanthat of the previous set with [3H]uridine-labeledvirus. This increased reduction may be due tothe loss of radioactivity from the polypeptidechains VP1 and VP2, which do not take part inthe attachment process. Approximately 1 logdifference was observed between the survivalcurve and penetration curve (Fig. 4), indicatingthat the impairment of viral coat protein maynot be the major cause of inactivation.

Effect of ozone ofviral RNA. To determinewhether viral RNA is damaged by exposure toozone, [3H]uridine-labeled poliovirus wastreated with 0.31-mg/liter residual ozone con-centration at pH 4.3 for 45 s. The RNA from thecontrol and ozone-treated samples were thenextracted and centrifuged on a 5 to 20% sucrosedensity gradient at 35,000 rpm for 210 min. Frac-tions collected from the density gradient analysiswere then assayed to determine the radioactivityassociated with each fraction. The results ofthese experiments are presented in Fig. 5A and5B.The sedimentation profile of the RNA ex-

tracted from the control virus (Fig. 5A) indicates

100

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CONTACT TIME, SECFIG. 4. Effect on poliovirus 1 penetration-attach-

ment to BGMhost cell and survival in a batch systemusing 0.51-mg/liter ozone residual concentration at20°C, pH 7.2. Viruses were labeled with "4C-aminoacid mixture. Percent survival was calculated as theratio of titers of the exposed viruses to that of controlvirus suspension.

Xi

II IIIII

.

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APPL. ENVIRON. MICROBIOL.

200- A (42 S)

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FRACTION NO.FIG. 5. Centrifugation analysis of[3Hluridine-la-

beledpoliovirus 1 RNA on a 5 to 20% sucrose densitygradient at35,000)rpm for210min. (A) RNA extractedfrom control virus suspension; (B) RNA extractedfrom ozonatedpoliovirus 1 suspension exposed to 0.3-mg/liter residual for 30 s atpH 4.3.

a single peak. The sedimentation coefficient ofthe RNA at the peak was calculated to be 42Sby the approximate method of Griffith (5). Thesedimentation coefficient of the RNA (42S) cor-responds well with the reported sedimentationcoefficient of poliovirus RNA (37S) in the liter-ature (17), suggesting that the peak obtained inthe sedimentation profile of these experimentsrepresented the single-stranded RNA of thepoliovirus. The sedimentation profile of RNA ofthe ozone-treated viruses (Fig. 5B) indicates thatthe viral RNA did not sediment in a single band;rather, two broad bands were observed. Thisobservation implies that the viral RNA was

damaged due to ozonation, possibly fragmentedinto a number of short chains.

DISCUSSIONInactivation of viruses is defined as their ina-

bility to replicate within host cells. Consideringthe structure of the enteric viruses, it is reason-able to assume that ozone causes physical dis-ruption of the particle or reacts with either theviral coat protein or the nucleic acid or both soas to affect attachment or intracellular step(s) invirus replication. Thus, barring physical disrup-tion, damage to an enteric virus particle could

affect: (i) extracellular reactions taking placebetween the host cells and the virus particlesleading to attachment and subsequent penetra-tion of viruses into the host cells; or (ii) intra-cellular biosynthesis taking place after viral ge-nome penetration into the host cell; or both.

Extracellular reactions are initiated when theinvading virus particle is adsorbed to the surfaceof a susceptible host cell. This probably involvesan interaction between the viral capsid proteinand the specific components of the host cellsurface. Rekosh (13), in a review article on mo-lecular biology of viruses, suggested that thesmall virus polypeptide VP4, present within thevirion, plays an important role in the adsorptionprocess. Crowell and Philipson (4) and Longer-Holm and Komat (8) demonstrated that bothcoxsackievirus and rhinovirus, which lack VP4,can be eluted from the host cells to which theyhad attached. Also, Cords et al. (3) and Breindland Koch (1) have reported that a loss in hostcell infectivity is associated with a loss of poly-peptide chain VP4 with both poliovirus andcoxsackievirus.

In studies designed to determine the mecha-nism of poliovirus inactivation by ozone, Riesseret al. (14) reported that upon reaction withozone, the capsid of the virus particle is dam-aged. They concluded that this, in turn, affectsthe attachment of the virus to a susceptible hostcell. However, their results indicated that al-though there was little change in percent pene-tration during the first 2 min of exposure toozone, there was a significant loss of viability,indicating that damage of the viral capsid mightnot be the major cause of viral inactivation byozone.Other research work relating to the effect of

ozone on compounds like proteins and nucleicacids is worthy of mentioning at this point, asthese are the two major constituents of a virusparticle. The reaction of ozone with amino acidsproteins has been reviewed by Mudd et al. (12).They reported that cysteine, methionine, tryp-tophan, tyrosine, histidine, cystine, and phenyl-alanine are oxidized by ozone, whereas otheramino acids are unaffected by ozone. The effectof ozone on nucleic acids was studied by Chris-tensen and Giese (2). Since absorbance at awavelength of 260 nm is approxrimately propor-tional to the concentration of nucleic acids, theyused this measurement to determine the effectof ozone on nucleic acid. After 15 s of exposureto ozone, the peak absorbance decreased to 50%of the control, and within 60 s absorbance at 260nm was undetectable.

In this study, inactivation experiments wereperformed in batch systems using radiolabeledpoliovirus 1 to investigate the mechanism of

722 ROY ET AL.

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ENTEROVIRAL INACTIVATION BY OZONE 723

inactivation by ozone. Exposure of viruses toozone did not result in their dissociation intomolecular subunits. Also, the viruses did notappear to form large aggregates after ozonation.Comparison of the electrophoretic profiles of

ozone-treated viruses with those of control vi-ruses revealed that two polypeptide chains ofthe viral capsid, VP1 and VP2, were damageddue to ozonation. The smallest viral polypeptidechain of the capsid that has been reported to beinvolved in attachment (VP4) was not signifi-cantly altered by ozonation. Attachment ofozone-exposed viruses was observed to be lessthan that of the control viruses. However, thedifference in percent attachment cannot accountfor the high percent inactivation that can beachieved for the same ozone dosage. Thus, itmay be concluded that the ability of viruses toattach to host cells is not significantly affectedas a result of exposure to a low residual concen-tration of ozone. This conclusion is consistentwith the fact that polypeptide chain VP4, whichhas been reported to be associated with attach-ment, was not affected as a result of ozonationin this study.By comparing the sedimentation profiles of

nucleic acids ofozone-exposed viruses with thoseof control viruses, we observed that viral nucleicacid was damaged as a result of ozonation. Itwould appear that the major cause of viral in-activation, as determined with poliovirus 1, byozone using a residual concentration less than0.3 mg/liter and a contact time up to 2 min isdamage ofthe RNA. This conclusion agrees withthe results of the kinetic studies (16), whichreported that the inactivation of viruses byozone is rate limited by the diffusional step ofozone through the protein coat into the nucleicacid core.

ACKNOWLEDGMENTSThis research was partially supported by the funds received

under an interagency agreement between the U.S. Army Med-ical Research and Development Command and the U.S. En-vironmental Protection Agency (DADA 17-72-C-2125, DAMD17-75-C-5006).We gratefully acknowledge the technical assistance pro-

vided by M. M. Soong, Department of Microbiology, Univer-sity of Mllinois, Urbana.

LITERATURE CTD1. Breindl, M., and G. Koch. 1972. Competence of sus-

pended HeLa cells for inactivation by inactivated polio-virus particles and by isolated viral RNA. Virology 48:136-144.

2. Christensen, E, and A. C. Giese. 1954. Changes inabsorption spectra of nucleic acids and their derivativesfollowing exposure to ozone and ultraviolet radiation.Arch. Biochem. Biophys. 51:208-216.

3. Cords, C. E., C. G. James, L. C. MeLaren. 1975. Alter-ations of capsid proteins of coxsackievirus A13 by lowionic strength. J. Virol. 15:244-252.

4. Croweil, R. L, and L Philipson. 1971. Specific altera-tions of coxsackie B3 eluted from HeLa cells. J. Virol.8:509-515.

5. Griffith, 0. M. 1976. Techniques of preparative, zonaland continuous flow ultracentrifugation. Beckman In-struments, Inc., Fullerton, Calif.

6. Katzenelson, E., B. Kletter, and H. I. Shuval. 1974.Inactivation kinetics of viruses and bacteria by use ofozone. J. Am. Water Works Assoc. 66:725-729.

7. Kuchler, R. 1976. Editor's comments on papers 17, 18and 19, p. 212-213. In R. Kuchler (ed.), Animal cellcultures and virology. Dowden, Hutchinson & Ross,Inc., Pa.

8. Longer-Holm, K., and B. D. Kornat. 1972. Early inter-action of rhinovirus with host-cells. J. Virol. 9:29-40.

9. Maizel, J. V., D. F. Summers, and M. D. Scharff. 1970.SDS-acrylamide gel electrophoresis and its applicationto the protein of poliovirus and adenovirus infectedhuman cells. J. Cell Physiol. 76:273-288.

10. Mandel, B. 1964. The extraction of ribonucleic acid frompoliovirus by treatment with sodium dodecyl sulfate.Virology 22:360-367.

11. Martin, E. M., and T. S. Work. 1962. Studies on proteinand nucleic acid metabolism in virus infected mamma-lian cells. Biochem. J. 83:574-582.

12. Mudd, J. B., R. Leavitt, A. Ongun, and T. T. Mc-Manus. 1969. Reaction of ozone with amino acids andproteins. Atmos. Environ. 3:669-882.

13. Rekosh, D. M. K. 1977. The molecular biology of picor-naviruses, p. 63-110. In D. P. Nayak (ed.), Molecularbiology of animal viruses, vol. 1. Marcel Dekker, Inc.,New York.

14. Riesser, V. M., J. R. Perrich, B. B. Silver, and J. R.McCammon. 1977. Possible mechanism of poliovirusinactivation by ozone, p. 186-192. In Forum on OzoneDisinfection, Proceedings of the International OzoneInstitute, International Ozone Institute, Syracuse, N.Y.

15. Rovozzo, G. C., and C. N. Burke. 1973. A manual ofbasic virological techniques. Prentice-Hall, Inc., Engle-wood Cliffs, N.J.

16. Roy, D., R. S. Engelbrecht, P. K. Y. Young, and E. S.K. Chian. 1980. Inactivation of enteroviruses by ozone.Prog. Water Technol. 12:819-36.

17. Ruckert, R. R. 1971. Picomaviral architecture. In E.Maramorosch and E. Kurstan (ed.), Comparative virol-ogy. Academic Press, Inc., New York.

18. Scarpino, P. V., G. Berg, S. L Chang, D. Dabling,and M. Lucas. 1972. A comparative study of the inac-tivation of vimses in water by chlorine. Water Res. 6:959-965.

19. Shechter, H. 1973. Spectrophotometric method for de-termination of ozone in aqueous solutions. Water Res.7:729-739.

20. Vrijsen, R., and A. Boeye. 1978. Gel electrophoresis ofproteindodecyl sulfate complexes in a pH gradient andimproved resolution of poliovirus polypeptides. Anal.Biochem. 85:355-366.

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