APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1980, p. 210-2180099-2240/80/01-0210/09$02.00/0

Vol. 39, No. 1

Mechanism of Ozone Inactivation of Bacteriophage f2CHI K. KIM, DAVID M. GENTILE, AND OTIS J. SPROUL*

Department of Civil Engineering, The Ohio State University, Columbus, Ohio 43210

The inactivation kinetics of bacteriophage f2 were studied by using ozone undercontrolled laboratory conditions. The phage were rapidly inactivated during thefirst 5 s of the reaction by 5 and 7 logs at ozone concentrations of 0.09 and 0.8 mg/liter, respectively. During the next 10 min, the phage were further inactivated ata slower rate in both treatments. The [3H]uridine-labeled f2 phage and itsribonucleic acid (RNA) were examined to elucidate the mechanism of ozoneinactivation, utilizing adsorption to host bacteria, sucrose density gradient anal-ysis, and electron microscopy. The specific adsorption of the phage was reducedby ozonation in the same pattern as plaque-forming unit reduction. RNA wasreleased from the phage particles during ozonation, although it had reducedinfectivity for spheroplasts. Electron microscopic examination showed that thephage coat was broken by ozonation into many protein subunit pieces and thatthe specific adsorption of the phage to host pili was inversely related to the extentof phage breakage. The RNA enclosed in the phage coat was inactivated less byozonation than were whole phage, but inactivated more than naked RNA. Thesefindings suggest that ozone breaks the protein capsid into subunits, liberatingRNA and disrupting adsorption to the host pili, and that the RNA may besecondarily sheared by a reduction with and/or without the coat protein mole-cules, which have been modified by ozonation.

The serious health concerns over the by-prod-ucts formed when chlorine is added to water fordisinfection have prompted a search for alter-natives. Ozone, one of the alternatives, has theadvantage offaster inactivation rates, but it doesnot have a persisting residual in water (2). Inaddition, its potential for formation of toxic by-products has not been evaluated completely (15,16). Inactivation kinetics with ozone have beenstudied actively in laboratory and pilot plantscales by using a variety of microorganisms,including viruses (11, 17, 29). Much informationattesting to the superiority of ozone over otherchemical disinfectants has accumulated (9, 30).Lower concentrations of ozone and shorter con-tact times are required compared with chlorineand other agents, and it is more effective thanother disinfectants against resistant organisms,such as amoebic cysts and viruses.Although the earlier all-or-none phenomenon

of ozone inactivation was explained as an ozonedemand exerted by the organisms themselves(22), recent reports indicate a threshold effect invirus ozonation (10, 13). Katzenelson and Bied-ermann (9) reported a two-stage inactivation byozone, in which 99% of the test organisms wereinactivated in 8 s and the remainder was inacti-vated only after several minutes of contact. Theydemonstrated that their two-stage kinetics couldbe attributed to virus aggregation. Limited in-formation is available on the mechanism of

ozone inactivation of bacteria and viruses, eventhough ozone has been used increasingly in thedisinfection of water.The ribonucleic acid (RNA)-containing bac-

teriophage f2, which is chemically and physi-cally very similar to enteric viruses, was firstintroduced in 1964 (8) as a model for inactivationstudies of the enteric viruses. Since then, thephage f2 has been used by many researchers (5,20, 21, 27) for the study of inactivation kineticsand inactivation mechanisms, using various dis-infecting agents. Hsu et al. (8) showed thatphage f2 RNA and poliovirus RNA were resist-ant to iodination and that inactivation of bothf2 and poliovirus were inhibited by increasingiodide ion concentrations. Phage f2 was used byOlivieri et al. (20) to study the mode of action ofchlorine, bromine, and iodine. They found thatthe mode of action depended upon the element.Chlorine inactivated naked f2 RNA at the samerate as it inactivated RNA in intact phage at pH7.5 or lower. The protein of the inactivatedphage was still able to adsorb to the host. Bro-mine inactivated naked RNA at the same rateas it inactivated intact phage, but the RNAprepared from bromine-treated virus was signif-icantly less inactivated than the intact virus.They suggested that the primary site of bromineinactivation was more likely to be the proteinmoiety of the virus. Iodine functioned throughiodination of the amino acid tyrosine in the

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OZONE INACTIVATION OF PHAGE f2 211

protein moiety of the phage and had almost noeffect on the nucleic acid.The inactivation mechanism of ozone has

been partially studied by using viruses and bac-teria. Using ozone in secondary effluent, Pavoniet al. (21) reported that the mechanism of de-struction of f2 phage and bacteria was probablyoxidative. Riesser et al. (25) reported that theprotein capsid of poliovirus type 2 was damagedby ozonation so that uptake into susceptible cellswas inhibited. Damage of the viral protein coatwas also demonstrated by DeMik and DeGroot(6), when bacteriophage 4X174 was exposed toair containing ozone. They also observed breaksin the phage deoxyribonucleic acid. Christensenand Giese (4) reported that the effect of ozoneon nucleic acids could be attributed to its actionon the purines and pyrimidines, each of whichappeared to be affected. In studies by Prat et al.(23) and Scott (26) with Escherichia coli deoxy-ribonucleic acid, the pyrimidine bases were mod-ified by ozonation, with thymine being moresensitive to ozone than cytosine and uracil.These reports provide limited information onthe mechanisms of ozone inactivation of virusesand especially on the effect of ozone on the viralRNA and the intact RNA and coat protein ofthe viruses.The bacteriophage f2 has been throught to be

an adequate model to study the inactivationmechanism of ozone because it consists of asingle-stranded RNA and two structural pro-teins having known amino acid sequences (31)and because it is inactivated by chlorine andother disinfectants in a manner similar to theenteric viruses (5). The objective of this studywas to determine the mechanism of the inacti-vation of bacteriophage f2 with ozone.

MATERIA1S AND METHODSPreparation, purification, and titration ofbac-

teriophage f2. Bacteriophage f2 (ATCC 15766-B)was propagated by using E. coli K-13 Hfr (ATCC15766) grown in tryptone-yeast extract broth mediumand the ammonium sulfate method described by Loeband Zinder (12), with minor modifications. These mod-ifications were to prepare 2 liters of host bacterialculture and to make the lysate by omitting the firstaddition of ammonium sulfate and omitting filtrationthrough a filter candle. The bacteriophage was furtherpurified by using cesium chloride at a concentration of0.65 g/ml and was centrifuged at a speed of 125,000 xg for 48 h in a Beckman L2-65B ultracentrifugeequipped with a Ti-75 rotor (Beckman Instruments,Inc., Palo Alto, Calif.). The phage band was colectedfrom the middle of the tube and dialyzed twice at 4°Cfor 24 h against 1 liter of 0.01 M phosphate buffercontaining 8.0 g of NaCl, pH 7.2, per liter. The dialy-sate was diluted with sterile triple-distilled water toabout 5.0 x 1012 plaque-forming units (PFU) per mland stored at -70°C until used. All of the phagesamples used in this study were titrated by using E.

coli K-13 as the host, according to the overlay methodof Adams (1).

Incorporation oftritium into bacteriophage f2.The RNA was labeled with [3H]uridine in generalaccordance with the method described by Oeschgerand Nathans (19). A 1-liter amount of Tris-pyruvate-glucose medium with low uridine (2.4 mg/liter) wasinoculated with 50 ml of overnight-grown E. coli K-12C-3000-38, which is a mutant requiring uridine, thy-mine, and the amino acids arginine, lysine, and histi-dine. After 24 h of incubation with aeration at 37°C,3 mCi of [3H]uridine (New England Nuclear Corp.,Boston, Mass.) was added. After an additional 30 minof incubation, the culture was infected with bacterio-phage f2 at a multiplicity of infection of 10 and incu-bated for another 24 h. The tritiated phage wereisolated by the method ofYamamoto and Alberts (32),using polyethylene glycol 6,000. They were furtherpurified by ultracentrifugation on cesium chloride,dialyzed, and stored in the manner described above.Preparation and assay of phage f2 infectious

RNA. Infectious RNA was prepared from bacterio-phage f2 by the phenol method described by Hof-schneider and Delius (7). The RNA isolated from 2 mlof tritiated phage was diluted to 2 ml with cold steriletriple-distiUed water. The RNA to be treated withozone was subsequently extracted six times with 2 mlof ethyl ether to remove traces of phenol. After thefinal extraction, the residual ether was removed bybubbling with nitrogen gas. AU of the RNA prepara-tion procedures were carried out in an ice bucket. Theinfectivities of the control and ozone-treated RNAswere assayed by using freshly prepared E. coli K-13spheroplasts as the hosts for the RNAs, according tothe method described by Hofschneider and Delius (7).

Ozonation of phage and RNA. The ozonationsystem used in this study is shown in Fig. 1. Ozonewas produced by a Linde model SG-4050 ozone gen-erator (Union Carbide Corp., South Plainfield, N. J.).The circulation system for the ozone was constructedwith stainless steel and Teflon tubing. A 2-liter boro-silicate glass bottle was used as the reactor. Ozone wasdissolved in 1 liter of sterile ozone demand-free 10-3M phosphate buffer containing 0.01 M NaCl, pH 7.0,at 25 ± 1°C. Ozone in the off gas was neutralized bypassage through a solution containing 500 g of sodiumthiosulfate and 10 g of potassium iodide per 3.78 litersof water. Ozone concentration was spectrophotomet-ricaUly measured with a Spectronic 20 spectrophotom-eter (Bausch & Lomb, Inc., Rochester, N.Y.) accordingto the method described by Shechter (28). AU theglassware used in this work was rendered ozone de-mand-free by soaking in an ozone solution (>2 mg/liter) for at least 1 h; it was then washed several timeswith distilled water, rinsed with triple-distilled water,and dried at 110°C for 5 h. The stock bacteriophageand RNA preparations were treated with an ozonesolution at a ratio of 1:100 in the reactor bottle or ina test tube at 25 ± 1°C. At appropriate intervals theozone-treated phage and RNA were transferred to asterile sodium thiosulfate solution (0.206 g/liter) hav-ing a volume of one-tenth the sample volume; thisimmediately neutralized the residual ozone. The con-trol phage and RNA samples were mixed with thephosphate buffer and sodium thiosulfate solution atthe same ratios as the ozone-treated samples. Sodium

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thiosulfate in the concentrations used did not affectthe sample titers.Host adsorption experiment. The procedures of

Brinton and Beer (3) were used to determine the lossof adsorption capability of ozonated viruses. E. coli K-15 T- is not a host for f2, and any adsorption would benonspecific, resulting from ozone-induced changes inthe phage. Samples (1 ml) of the control and ozone-treated phage were mixed with 1 ml of E. coli K-13and E. coli K-15 T- in tryptone-yeast extract brothmedium (5 x 107 cells per ml) for 10 min at roomtemperature. This mixture and a phage control werefiltered at a vacuum of 10 inches (25.4 cm) of mercuryand were washed four times with 2 ml of tryptone-yeast extract medium through a 0.45-,m membranefilter (type HA; Millipore Corp., Bedford, Mass.)which had been pretreated with 1 ml of 3% bovineserum albumin. The filter membranes were dried for15 min at 85°C and were then placed in 10 ml ofLiquifluor cocktail (New England Nuclear Corp.), andthe radioactivity was counted for 5 min with a PackardTri-Carb model 3375 liquid scintillation spectrometer(Packard Instrument Co., Inc., Downers Grove, Ill.).

Electron microscopy. Control and ozone-treatedviruses were observed with an electron microscopealone or after mixing with host bacteria. Portions (1ml) of the control (5 x 109 PFU/ml) and ozone-treatedphage were mixed with 1 ml of E. coli K-13 (5 x 106cells per ml) for 10 min at room temperature. Onedrop of a mixture was put on a Formvar- and carbon-coated 300-mesh copper grid (Ladd Research Indus-tries Inc., Burlington, Vt.) and dried for 30 min. Excesswater was drained with filter paper, and the virusesand bacteria were negatively stained with 2% uranylacetate for 3 min. After the excess stain was drainedoff and after air drying, the grids were examined witha Phillips EM 300 electron microscope (Phillips Elec-tronics, Cincinnati, Ohio) at an accelerating voltage of80 kV.

Sucrose density gradient analysis. Sucrose gra-

dients were made with 5 to 17% or 5 to 20% sucrose in0.01 M phosphate buffer containing 8.0 g of NaCl, pH7.2, per liter. Portions (1 ml) of the control or ozone-treated phage and RNA samples which had beenlabeled with [3H]uridine were put on top of an 11-mlgradient and centrifuged at 110,000 or 149,000 x g for4 or 8 h at 4°C with a Beckman L2-65B ultracentrifuge,using an SW41 rotor (Beckman Instruments, Inc.).The gradient was fractionated by collecting 15, 25, or50 drops for each fraction with an ISCO model 180density gradient fractionator (Instrumentation Spe-cialties Co. Inc., Lincoln, Nebr.), using a 40% sucrosesolution. Each fraction was examined for refractiveindex, infectivity of the phage, and radioactivity. Thephage in the fractions were titrated after dialyzingagainst 0.01 M phosphate buffer containing 8.0 g ofNaCl, pH 7.2, per liter at 4°C overnight. A 10-mlportion of Biofluor cocktail (New England NuclearCorp.) was mixed with each fraction, and radioactivitywas counted in the way described above.

RESULTSThe ozone inactivation kinetics of bacterio-

phage f2 are shown in Table 1. A rapid phageinactivation during the first 5 s of exposure wasnoted, with 5 logs lost at 0.09 mg of ozone perliter and more than 7 logs lost at 0.8 mg of ozoneper liter. During the next 10 min, the phage weregradually inactivated at a slower rate in bothozone concentrations. The specific and nonspe-cific adsorptions of the phage samples examinedby the filtration method are shown in Table 1.The specific adsorption of the phage to the hostE. coli K-13 gradually decreased at a slower ratein a 0.09-mg/liter ozone solution and at a fasterrate in a 0.8-mg/liter ozone solution. For bothtreatments the general trends of reduction in thespecific adsorption of the phage were faster for

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OZONE INACTIVATION OF PHAGE f2 213

TABLE 1. Ozone inactivation kinetics ofbacteriophage f2 and its adsorption to host andnonhost bacteria and to a cellulose nitrate filter

Adsorption of f2 phageReac- Residual Survival of (cpm) to:ation ozone rtinme (mg/li- (PFU/mg) E. coli E. coli Filter(s) ter) KPFUm3)K- mem-

K-13 15T- brane

0 0.09 5.3 x 109 2,617 53 1395 _b 8.7 x 1i 1,742 103 8810 0.086 6.2 x 104 1,777 122 7330 0.085 3.3 x 103 1,660 170 8160 0.083 5.6 x 102 1,363 101 84120 0.08 5.0 x 102 1,457 144 77600 0.065 4.6 x 101 1,199 165 67

0 0.80 5.3 X 109 2,617 53 1395 - 2.6 X 103 1,013 185 7910 0.75 6.8 x 102 851 103 7430 0.68 5.2 x 102 562 89 6960 0.64 2.0 X 100 434 74 66120 0.53 1.0 x 100 372 61 57600 0.30 0 283 52 58

aE. coli K-13 and E. coli K-15 T- were used as thehost and nonhost bacteria, respectively, of phage f2. A0.45-,m Millipore type HA membrane filter was usedfor nonspecific adsorption of the phages.b, Not determined.

the first 5 s and slower for the next 10 min andwere similar to the reduction in PFU. On theother hand, nonspecific adsorptions of the phageto the nonhost female strain E. coli K-15 T- andto a cellulose nitrate filter membrane were notsignificantly changed by ozonation at eitherozone concentration.The untreated control phage and the phage

samples which had been treated with 0.09 mg ofozone per liter for 5 s (referred to as light treat-ment) and 0.8 mg of ozone per liter for 30 s(referred to as heavy treatment) were sedi-mented in a 5 to 20% sucrose gradient and frac-tionated by collecting 25 drops for each fraction.On the other hand, phage f2 RNA was firstextracted from the stock phage and then lightlyor heavily treated with ozone. The naked RNAsamples were sedimented and fractionated inthe same manner as the phage samples. ThePFU of the phage and the radioactivities of thephage and naked RNA are shown in Fig. 2. Thepeaks of the PFU curves in the control andozone-treated phage were found in the mixtureof fractions 9 and 10 in all three samples; lowernumbers of PFUs were found in the sampleswhich had been treated at the heavier concen-tration of ozone for a longer time. However, theradioactivity (presence of f2 RNA) in the ozone-treated phage was localized in the upper frac-tions, which had lower densities. The radioactiv-

ity of the control phage was found mainly in thesame gradient fractions in which the peak of thePFU curve was seen (Fig. 2A), but the radioac-tivity in the lightly and heavily treated phagesamples was observed in fractions 3 and 4 andfractions 2 and 3, respectively (Fig. 2B and C).The radioactivities of the control naked RNAand the ozone-treated naked RNA samples werenearly the same; their peaks were in fractions 2and 3 in all three samples. The distribution ofheavily treated naked radioactive RNA wasidentical to that of the heavily treated phagesample (Fig. 2C), thereby indicating that theRNA from the ozonated phage had been liber-ated into the water.To prove leakage of RNA from the phage

during ozonation and to correlate the specificadsorption of ozone-treated phage with the in-activation rate of the phage, the control andozone-treated phage were mixed with host bac-teria and observed with an electron microscope(Fig. 3). The control phage had the integrity ofthe icosahedral structure and attached to pili ofthe host (Fig. 3A). The phage unadsorbed to thepili were scattered over the grid as single parti-cles. A few broken particles were also observedin the control sample. In the phage sampleswhich had been lightly treated with ozone, somebroken phage particles were seen, and otherintact phage were attached to the pili (Fig. 3B).Almost all of the phage were disrupted in theheavily treated sample (Fig. 3C), and only a fewintact phage were found in the background orattached to the pili. Many subunits of the pro-tein capsid were observed in the heavily treatedphage sample (Fig. 3C).The survival fractions of the phage and naked

RNA samples were examined after ozonationand compared with the RNA extracted from thephage previously treated with ozone (Fig. 4). Inthe case of the light ozone treatment, the RNAextracted from the ozone-treated phage (about5 logs of inactivation) showed about 3 logs ofinactivation, but the separately treated nakedRNA lost less than 1 log during ozonation (Fig.4A). In the heavily treated sample, the phage,the RNA extracted from the treated phage, andthe naked RNA showed, respectively, more than7, 5, and 2 logs of inactivation (Fig. 4B). In bothtreatments the naked RNA was much less in-activated by ozonation than the RNA inside thephage particle.To examine any difference in inactivation

rates between naked RNA and RNA enclosed inthe phage coat, the ozone-treated and untreatedsamples of both RNAs were sedimented in 5 to17% sucrose gradients and fractionated by col-lecting 15 drops for each fraction. The sucrose

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214 KIM, GENTILE, AND SPROUL

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FIG. 2. Sucrose density gradient analysis of untreated control bacteriophage f2 (A) and samples whichwere treated with 0.09 mg ofozoneper liter for 5 s (B) and with 0.8 mg ofozoneper liter for 30 s (C). The nakedRNA which had been extracted from the stock phage was ozonated in the same manner as the phage. Threeseparate gradients were used to measure the radioactivities ofphage and naked RNA andphage infectivity.Both samples ofphage and naked RNA were centrifuged at 110,000 x g for 4 h in a 5 to 20% sucrose gradient.For each fraction 25 drops was collected from the top to analyze the radioactivity of the phage (0) and thenaked RNA (A). For each fraction 50 drops was collected for titration ofphage infectivity (U). The peaks ofPFUs remained in the mixture of fractions 9 and 10 after ozonation, whereas the radioactivity peaks of thesamples were moved up by ozonation. The radioactivities of all naked RNA samples were essentiallyunchanged, showing theirpeaks in fraction 3. Note a minor radioactivity peak of the controlphage sample infraction 3.

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FIG. 3. Electron micrographs of control phage f2 (A) andphage which were treated with 0.09 mg of ozoneper liter for 5 s (B) and with 0.8 mg of ozone per liter for 30 s (C). The phage samples adsorbed to the hostbacteria were stained with 2% uranyl acetate. The control sample showed that almost all of the phage wereintact and that many ofthem adsorbed to the host pili. A few ofthem are seen partially damaged. Some ofthelightly treated phage were still intact and adsorbed to the pili but others were broken down to larger piecesofprotein capsid. Most of the heavily treated phage were broken down to many subunits of the capsid, andonly a few of them can be seen intact and adsorbed to the pili or unadsorbed. Many subunits (arrows) of theprotein capsid are seen all over the grid. Bars = 0.1 ,um.

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216 KIM, GENTILE, AND SPROUL

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FIG. 4. Comparative survival fractions ofphage f2(a), RNA extracted from phage previously treatedwith ozone (a), and naked RNA (A). The sampleswere treated with 0.09 mg ofozone per liter for 5 s (A)and with 0.8 mg of ozone per liter for 30 s (B). TheRNA extracted from the ozone-treated phage wasless inactivated than thephage and more inactivatedthan the naked RNA in both treatments.

density gradient analysis is shown in Fig. 5. Inthe naked RNA samples (Fig. 5A), the sedimen-tation velocity of the ozone-treated naked RNAwas slightly changed, and the RNA showed ra-dioactivity peaks in the upper fractions, whichhad lower buoyant densities. On the other hand,the RNA extracted from the phage previouslytreated with ozone was sedimented at a slowerrate after ozonation than the naked RNA was(Fig. 5B). The counts per minute were signifi-cantly reduced in the RNA samples extractedfrom the previously ozonated phages (Fig. 5B).The differences in the change of sedimentationvelocities between the naked RNA and the RNAextracted from the previously ozone-treatedphage were in agreement with the results shownin Fig. 4, indicating that the naked RNA wasless affected by ozone than the RNA in theintact phage. The radioactivity distributions ofboth groups ofRNA after ozonation were shiftedtoward fractions with lower densities, dependingupon the ozone concentration and length of thereaction.

DISCUSSIONA small amount of organic matter in a solution

can cause the rapid dissipation of ozone anddecreased inactivation rates of test organisms.To meet this demand, the ozonation system usedin this study was carefully constructed with

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FIG. 5. Sucrose density gradient analysis of ozon-ated naked RNA (A) and RNA extracted from thephage previously treated with ozone (B). The controlRNA (0) and the RNA which had been treated with0.09 mg of ozone per liter for 5 s (U) and with 0.8 mgof ozone per liter for 30 s (A) were centrifuged in a 5to 17%o sucrose gradient at 149,000 x g for 8 h and 15drops for each fraction were collected from the top.The RNA extracted from the ozone-treated phage isseen in the upper fractions, which have a lower buoy-ant density and are lower in counts per minute thanthe naked RNA in both treatments.

stainless steel and Teflon tubing, and the systemwas calibrated to produce ozone concentrationsas low as 0.01 mg/liter. All glassware and solu-tions for this study were made ozone demand-free. The ozone reactors were closed during themixing of the samples, and the residual ozonewas evaluated with its natural decomposition,which occurred during the contact period.

Inactivation of bacteriophage f2 was demon-strated during this study by a quick reactionwith a low concentration of ozone, as suggestedby Venosa (30). Only 5 s was needed to inactivate5 logs of the phage with 0.09 mg of ozone perliter, but a longer time was required to com-pletely inactivate the phage (10 min with 0.8 mg

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OZONE INACTIVATION OF PHAGE f2 217

of ozone per liter). The inactivation kinetics ofthe phage revealed a two-stage action of theozone. Katzenelson et al. (10) thought that theirtwo-stage phenomenon was due to clumping of0.5 to 1.0% of their poliovirus 1. There is evidence(33) that aggregation of some enteric viruses canreduce the inactivation rate by protecting theviruses from contact with disinfectants. Thephage were stored at -70°C and thawed justbefore use in order to reduce any possiblechanges in their sensitivity to ozone, as sug-gested by Katzenelson and Biedermann (9). Inthe f2 phage stock as well as in the diluted phagesamples used in this study, aggregation of thephages was not detected by electron microscopy.The phage were widely distributed through-

out the sucrose gradient after sedimentation ofboth the ozone-treated and untreated phagesamples. At a concentration of about 104 PFU/ml or less, the radioactivity was too low to bedetermined in counts per minute. This can beexplained by the calculation of 7.9 x 105 PFU/cpm in the fraction of the control phage whichshowed peaks in both PFU and radioactivity. Aminor peak in fraction 3 of the control phagewas thought to be RNA, since a similar peakwas observed in the gradient of the naked RNAsample which had been used as a reference.Leakage of the RNA from some phage maypossibly occur during the thawing and dilutionprocedures of the phage stock, and electron mi-crographs of the control sample revealed a fewdamaged phage particles.The electron micrographs ofthe ozone-treated

phage showed breakdown and alteration of thephage particles. The extent of the damage wasproportional to the concentration of ozone andthe period of reaction time. The lightly treatedphage showed some deformed particles and largepieces of broken capsids, whereas uniform sizesubunit particles were observed in the heavilytreated phages. These subunits resemble the 11S subunits of bacteriophages f2 (34) and R17(18) which were obtained by treating the phageswith 4M guanidine hydrochloride at neutral pH.Damage of the protein coat by ozonation hasbeen reported in both RNA-containing polio-virus (25) and deoxyribonucleic acid-containingbacteriophage OXX174 (6). These changes are alsosupported by the absorbance changes of theproteins and amino acids (14) when treated withozone. Mudd et al. reported that proteins wereinactivated directly by reaction of ozone withsusceptible amino acid residues and that cys-teine, tryptophan, and methionine were the mostsusceptible among the amino acids. Tryptophanand methionine were also shown (24) to be par-ticularly sensitive to ozone. The breakdown off2 phage by ozonation can thus be explained

since the coat proteins contain cysteine, trypto-phan, and methionine.About 106 PFU of infectious RNA per ml was

recovered by phenol extraction from about 1010PFU of phage per ml. The infectious RNA wasevaluated for the presence of the phage withintact host bacteria, and no plaques were pro-duced. Both naked RNA and RNA isolated fromphage previously treated with ozone were pre-pared from the same number of phage and di-luted to react with ozone under the same con-ditions so that the same volume for the finalpreparation of the RNA could be directly com-pared for titration of infectivity and radioactiv-ity. The phenol-extracted RNA was isolated byprecipitation with ethanol and centrifugation.The decrease in radioactivity in the RNA sam-ples isolated from the phage previously treatedwith ozone may be attributed to the possibilitythat the RNA sheared during ozonation was notrecovered in this step. It is not clear whetherozone shears the RNA directly or by a secondaryreaction involving other factors. However, thelatter seems more likely since the changes inradioactivity peaks and counts in Fig. 5B weremore drastic than those in Fig. 5A. Few reportsare available on the effect of ozone on RNA.Christensen and Giese (4) and Scott (26) re-ported that both purines and pyrimidines werechanged in ultraviolet absorbancy by ozonation.Pyrimidine bases also react with ozone (4, 23),with thymine being more susceptible to ozonethan cytosine or uracil. If it is true that amongthe pyrimidine bases uracil is not affected byozone, then f2 phage RNA should be more re-sistant to ozone than deoxyribonucleic acid.The results obtained in this study show that

ozone breaks the protein capsid of phage f2 intomany subunits, liberating RNA into the solutionand disrupting adsorption to the host pili. TheRNA in the intact phage was less inactivated byozonation than were whole phage, but moreinactivated than naked RNA. This suggests thatthe coat protein may be involved in the inacti-vation of the RNA, probably by a secondaryreaction of the RNA with the protein moleculesmodified by ozonation. The RNAs extractedfrom f2 phage before and after ozone treatmentretained their infectivity to the spheroplastsafter ozonation despite some reduction. Our re-sults suggest that the RNAs of enteric virusesmay retain their infectivities after liberationfrom the viral particles during ozonation ofwaterand wastewater, if they are inactivated by ozonein the same manner as f2 phage.

ACKNOWLEDGMENTSThe late David M. Gentile initiated this work while a Ph.D.

candidate at the University of Maine. His foresight and wis-

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dom in developing the ideas subsequently supported by theobserved data are acknowledged. The Union Carbide Corp.graciously loaned the ozonator for this work.

This work was supported in part by funds provided by theU.S. Department of the Interior Office of Water Research andTechnology under project B-013-ME, as authorized by theWater Research and Development Act of 1978.

LITERATURE CITED

1. Adams, M. H. 1959. Bacteriophages. Interscience Pub-lishers, Inc., New York.

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3. Brinton, C. C., Jr., and H. Beer. 1967. The interactionof male-specific bacteriophage with F pili, p. 251-289.In J. S. Colter and W. Paranchych (ed.), The molecularbiology of viruses. Academic Press Inc., New York.

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