Effects of ozone treatment on the infectivity of hepatitis A virus JAMES M. VAUGHN' Department of Microbiology, University of New England College of Medicine, Biddeford, ME 04005, U.S.A.

Yu-SHIAWCHEN Department of Microbiology, SUNY School of Medicine, Stony Brook, NY 11794, U.S.A. AND

JAMES F. NOVOTNY AND DEBORAH STROUT'

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Received January 8, 1990 Accepted May 10, 1990

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Department of Microbiology, University of New England College of Medicine, Biddeford, ME 04005, U.S.A.

VAUGHN,J. M., CHEN,Y.-S., NOVOTNY, J. F., and STROUT, D. 1990. Effects of ozone treatment on the infectivity of hepatitis A virus. Can. J. Microbiol. 36: 557-560. The inactivation of a large-focus-forming variant of hepatitis A virus (HM-175) by ozone was investigated. Experiments using mainly single-particle virus preparations suspended in phosphate-carbonate buffer were conducted over a range of pH levels (6-8) at 4°C. Viral enumerations involved the use of a radioimmunofocus assay. While some tolerance to lower (i.e., 0.1-0.5 mg/L) ozone residuals was noted, the exposure of virus particles to ozone concentrations of I mg/L or greater at all pH levels resulted in their complete (5 log) inactivation within 60 s. The pH-related effects that were observed were not considered to be significant. Key words: hepatitis A virus, ozone, disinfection. VAUGHN,J. M., CHEN,Y.-S., NOVOTNY, J. F., et STROUT, D. 1990. Effects of ozone treatment on the infectivity of hepatitis A virus. Can. J. Microbiol. 36 : 557-560. Nous avons CtudiC le pouvoir inactivant de I'ozone sur un variant du virus de I'hCpatite A (HM-175) qui formait une grosse zone lors d'Cpreuves de focalisation. En utilisant des prkparations contenant principalement des particules virales isolCes en suspension dans un tampon phosphate-carbonate, on a fait des essais a 4°C a diffkrents pH (6-8). Les numkrations virales ont CtC faites par une mCthode de radioimmunofocalisation. Malgrk une certaine tolkrance des faibles niveaux d'ozone rksiduel (c.-a-d., 0,l-0,5 mg/L), le contact des particules virales avec des concentrations d'ozone Cgales ou supkrieures A 1 mg/L, et tous les pH vkrifiks, a produit I'inactivation complkte (5 log) en moins de 60 s. Des effets reliks au pH ont kt6 observks mais pas de f a ~ o nsignificative. Mots clks : virus de I'hCpatite A, ozone, dksinfection. [Traduit par la revue]

Introduction Hepatitis type A virus (HAV), a picornavirus now classified as enterovirus type 7 2 (Melnick 1982), was one of the earliest to be associated with transmission by the water route (Mason and McLean 1962; Cliver 1966; Mosley 1967). Although overshadowed in frequency by outbreaks of acute nonbacterial gastroenteritis (V. J. Cabelli. 1982. Proceedings of the Symposium on Viruses and Disinfection of Water and Wastewater, 1-4 September 1982, University of Surrey, U.K. University of Surrey Press, U.K. pp. 107-130), the severity of the disease, along with its explosive nature, qualifies its consideration as the most serious waterborne viral agent. During the period 1920- 1983, nearly 80 major outbreaks reported in the United States were linked to the ingestion of wastewater-contaminated drinking water (Craun 1986a, 1986b). In spite of its prominence as an environmental contaminant, the difficulties encountered in the isolation, in vitro propagation, and assay of HAV have hampered attempts to study its response to inactivation with chemical disinfectants. As a result, relatively few published reports of disinfectant-induced HAV inactivation rates are currently available. To date, HAV inactivation data have been reported for such agents as chlorine (Neefe et al. 1947; Peterson et al. 1983; Grabow et al. 1983), glutaraldehyde (Passagot et al. 1987), iodine (Coulepis et al. 1980), and a variety of antiviral substances including protarnine, 'Author to whom correspondence should be addressed. 'Present address: Analytab Products Incorporated, Plainview, NY 11803, U.S.A. Printed in Canada i Imprime au Canada

atropine, selenocystamine, taxifolin, and catechin (Biziagos et al. 1987). As of this writing, only two published studies have addressed HAV inactivation by ozone (Botzenhart and Herbold 1988; Herbold et al. 1989), a prominent alternative to chlorine for the disinfection of water and wastewater (Venosa 1983). Recent advances in methods for in vitro cultivation and quantitation of HAV (Provost et al. 1979; Daemer et al. 198 1 ; Lemon et al. 1983) have greatly facilitated the investigation of HAV inactivation dynamics. These methods were adapted for use in an evaluation of the effects of ozone concentration, exposure time, and pH of the ozone-buffer solutions on the infectivity of a purified, large-focus-forming variant of hepatitis A virus (HM-175).Studies were conducted at pH 6 . 0 , 7 . 0 , and 8.0.

Materials and methods Virus and host cells BS-C-1 cells, obtained from Dr. Mark Sobsey, University of North Carolina, Chapel Hill, were routinely propagated in Eagle's minimum essential medium (EMEM) supplemented with antibiotics, glutarnine, nonessential amino acids, and 10% fetal calf serum. Hepatitis A virus (HM-175) was also received from Dr. Sobsey. The virus was serially passaged for 5-18 weeks in our laboratory on monolayers of BS-C-1 cells grown in 800-cm2 roller bottles to select for a rapidly growing, large focus-forming variant as described by Cromeans et al. (1987). Use of this variant significantly reduced the incubation times required for the production of the virus stock and the conduct of assay procedures used in this study. Cells harvested from roller bottles were chilled to 4'C, pelleted (4000 X g, 10 rnin), washed twice with phosphate-buffered saline (PBS), resuspended in 5 rnL PBS, and

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freeze-thawed three times in ethanol - dry ice. Following the addition of 10 mL PBS, the cells were sonicated (20 Hz for 15-20 s; Kontes Microultrasonic Cell Disrupter) and extracted four times with chloroform. The resulting aqueous phases were combined and the viruses were pelleted by ultracentrifugation through 20% sucrose (Beckman L5-75 preparative ultracentrifuge, 185 000 X g, 10 min). The pellets were then resuspended in PBS, sonicated, and 2-mL volumes were layered onto sucrose cushions containing 1 mL of 50%, and 2 mL of 20% sucrose. These tubes were then centrifuged for 20 min at 100 000 x g (20°C) and the top 2.5 mL of each was pooled and stored at 4OC. This latter procedure, first reported by Young and Sharp (1977) using poliovirus, favored the generation of stocks of HAV, which having a buoyant density similar to that of poliovirus (Siege1 and Frosner 1978), contained mostly single particles (it must be assumed, however, that small viral aggregates were also present). Prior to each experiment, the virus stock was extensively dialyzed against ozone-demand-free (ODF) PBS (pH 7.0) to remove residual sucrose present in the centrifuge cushions.

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Virus assay HAV was assayed on monolayers of BS-C-1 cells using a radioimrnunofocus assay (RIFA; Lemon etal. 1983). Briefly, virus samples were diluted in Tris-buffered saline (TBS, pH 7.0) and inoculated on to duplicate host cell monolayers in 60-cm acetone-resistant petri dishes. Following a 2-h adsorption period, the inocula were decanted and replaced with 5 mL of overlay medium containing EMEM and agarose (SeaKem LE, FMC BioProducts). The dishes were then incubated (36"C, 5% COz) for 5 days, refed (1-2 mL EMEM-agarose), and returned to the incubator for an additional 3 days. At harvest, the agarose layers were carefully removed, and the cell sheets were washed, fixed with acetone, inoculated with 1 mL '25~-labeledIgG anti-HAV antibody (300 000 - 500 000 cpm; Abbott Laboratories) and incubated for 4 h. The antiserum was then aspirated and the monolayers were washed (five times) with PBS. The petri dish bottoms, containing the treated cell monolayers, were cut out and mounted with X-ray film (Kodak X-AR5) in an 8 X 10 in. (1 in. = 25.4 mm) cassette equipped with enhancer screens. The cassettes were maintained at -70°C for 4-5 days to assure maximum exposure. Films were then developed, and infectious foci were counted and tabulated as radioimmunofocusforming units (rfu) per unit volume. Experimental procedure Prior to each experiment, ozone stock solutions were prepared in ODF phosphate-carbonate buffer (Katzenelson et al. 1979) at the desired pH and ionic strength using the formulations of Sharp and Leong (1980). Ozone was generated from a Welsbach ozonator operating at 80 V. Dissolved ozone concentrations were determined by the spectrophotometric method of Schecter (1973). All experiments were carried out at 4OC as previously described by Vaughn et al. (1987). Briefly, 100-mL aliquots of ozone-treated buffer were inoculated with 1 mL of dialyzed virus stock (-lo6-10' rfu/mL) and gently mixed on a magnetic stirrer. Sample aliquots (10 mL) were collected at intervals and placed in test tubes containing 0.1 mL of 0.5 M sodium thiosulfate to reduce the ozone. Ozone residuals at the end of each experiment were measured to assess dissipation levels during the time of each study. Zero time control experiments (to determine initial virus concentrations) were carried out in 100-mL volumes of ODF buffer that had been inoculated with 1 mL of the virus stock. All samples were then treated with 0.5 mL of chloroform to inactivate microbial contaminants, diluted in TBS, and assayed for virus in BS-C-1 cells by the RIFA procedure. Positive and negative HAV controls were used in each assay to verify host cell susceptibility and the absence of endogenous HAV contamination. Each experiment was repeated three times to determine variability. Statistical analyses were performed on a Macintosh SE computer using preprogrammed software.

Results Ozone concentrations in stock solutions maintained at 4°C were stable for 5 to 6 min. Ozone dissipation observed during

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Time ( s )

FIG. 1. Effect of ozone treatment on the infectivity of HAV at (A) pH 6.0, (B) pH 7.0, and (C) pH 8.0. Nt/No, number of infectious foci at any time / number of foci at zero time. Ozone concentration (mg/mL): A, 0.1; A, 0.5; 1.0; 0, 2.0.

the course of each experiment (180 s) averaged 0.1 mg/L. All ozone residuals reported represent those measured at the initiation of each experiment. Datum points on each curve are median values from three separate experimental trials. The results of inactivation studies are presented in Figs. 1A1C. HAV was quite tolerant to an ozone residual of 0.1 mg/L at all pH levels tested. At pH 6.0 (Fig. 1A) , appreciable decreases in HAV titer were not noted until after 120 s of exposure, with subsequent decrease to approximately 1 log below that of the input level requiring an additional 60 s of exposure. Inactivation efficiencies were even more diminished at neutral and alkaline pH (Figs. 1B and 1C). Inactivation of HAV by 0.5 mg/L ozone residuals was substantially enhanced at all pH levels. An initial 1.5 log decrease in titer was observed during the first 30 s of exposure at pH 6.0. During the next 90 s, however, the virus concentration remained relatively constant. At pH 7.0, a 3 log decrease in titer occurred at a logarithmic rate over the course of the 180-s exposure period. A similar level of inactivation was

VAUGHN ET AL.

TABLE1. Approximate times for 99.99%inactivation of hepatitis A virus by ozone

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pH Ozone concn. (mg/L) 99.99% inactivation(s)

"90% reduction at -165 s. b90%reduction at -35 s. '90% reduction at > 180 s. d90%reduction at -60 s. '90% reduction at -65 s.

observed at pH 8.0, with the exception that only 2 logs of input HAV were removed during the exposure cycle. A significant improvement in viricidal activity was observed when ozone concentrations were increased to 1.0 mg/L. A 5 log decrease in HAV infectivity occurred within 30 s at pH 6.0 and 7.0 and within 60 s at pH 8.0. It was also noted that when the ozone concentrations in pH 8.0 experiments were increased to 2.0 mg/L, 5 log inactivation occurred within 30 s. Four log (99.99%) HAV inactivation times were derived by extending a line through the y-axis (N,INo, number of virus foci at any time 1 number of foci at time zero) to its point of intersection on each inactivation curve, and then vertically to its corresponding point on the x-axis. Inactivation data were compared on the basis of disinfectant concentration and pH level. The resulting figures, presented in Table 1, delineate both the relative resistance of HAV to the lower ozone concentrations and the modest influence of increasing alkalinity on virus stability.

Discussion The effects of ozone treatment on the infectivity of various human viruses, principally enteroviruses and rotaviruses, have been addressed in a number of laboratory investigations. Studies conducted in a variety of liquid media ranging from distilled water to wastewater effluents and ambient surface waters have demonstrated the rapid and highly efficient inactivation of these viruses with relatively low ozone residuals (Majumdar et al. 1973; Burleson et al. 1975; Katzenelson et al. 1979; Roy et al. 1981; Vaughn et al. 1987). In two reports of HAV inactivation by ozone from the same laboratory (Botzenhart and Herbold 1988; Herbold et al. 1989), unpurified HAV stock (HAVIHFSIGBM), along with poliovirus type 1, were suspended in PBS and treated with ozone under conditions of constant flow. Virus enumerations involved a 50% tissue culture infectious dose method (TCIDSO;Karber 1931). The authors reported significantly differing patterns of inactivation between the two virus types, with HAV requiring nearly three times the ozone residual to effect a complete inactivation (Herbold et al. 1989). Inactivation was more efficient at lower temperatures, with 4 log infectivity reductions occurring within 8 s at a constant ozone concentration of 0.1 mg/L at 10°C, versus a 0.22 mg1L residual required to achieve the same inactivation

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efficiency at 20°C. The authors indicated that a disinfectant concentration of 0.38 mg/L would be sufficient to instantaneously inactivate all of the input virus at the higher temperature. In the present study, purified HAV stocks consisting primarily of single particles and small aggregates, suspended in an ionically defined buffer, were exposed to various ozone concentrations and pH levels at 4°C. HAV enumeration was based upon the quantitation of infectious foci on host cell monolayers. The HAV variant used in this study appeared to be somewhat more resistant than the strain employed in the articles recounted above. The rapid infectivity reductions resulting from treatment with residuals of 0.1 to 0.2 mg/L in the earlier work were not encountered in the present experimental series until 1.0 mg/L ozone concentrations had been applied. The apparent disparity between these results and those of the earlier studies may reflect differences between HAV strains used, the relative sensitivities of the virus detection methods, and dissimilarities in the experimental procedures and materials used in each. Specific rationale notwithstanding, the present findings illustrate the need to consider the use of ozone residuals that are higher than the 0.4 mg/L concentration proposed by some researchers for the effective disinfection of drinking water (Block et al. 1981). As the continued use of chlorine for the disinfection of potable water supplies comes under greater scrutiny owing to the potential health hazards posed by the resulting chlorinated hydrocarbons (Wang et al. 1978; Schnoor et al. 1979), the need for a safe and efficient alternative becomes more critical. Considerable attention has been focused on the use of ozone, regarded by many to be less hazardous and more effective than chlorine (Venosa 1983; National Academy of Sciences. 1979. Report of the subcommittee on efficacy of disinfection of the Sale Drinking Water Committee to the U.S. EPA. National Academy of Sciences, Washington, DC). One measure of the overall utility of ozone is its potential as a virucide. Studies to date have documented the sensitivity of a variety of potentially waterborne viral agents to treatment with ozone (Kessel et al. 1943; Burleson et al. 1975; Majumdar et al. 1973; Farooq and Akhlanque 1983; Katzenelson et al. 1979; Vaughn et al. 1987; J . F. Snyder and P. W. Chang. 1974. Proceedings of the I01 Workshop on Aquatic Applications of Ozone, September 1920, Boston, MA. International Ozone Institute, Boston. pp. 82-99). Results of the present study, along with those of Botzenhart and Herbold (1988) and Herbold et al. (1989) demonstrate the effectiveness of this disinfectant in the inactivation of hepatitis A virus, one of the most potent waterborne pathogens.

Acknowledgments This research was supported by grant R-812140-01 from the U.S. Environmental Protection Agency, Dr. Donald Carey, Project Officer. The authors acknowledge the efforts of Lois J. Baranosky and Timothy Strout in the conduct of this research. BIZIAWS, E., CRANCE, J. M., PASSAWT, J., ~ ~ D E L O I N R.C1987. E, Efficiency of antiviral substances on hepatitis A virus replication in vitro. J. Med. Virol. 22: 57-66. J. C., RICHARD, Y., HARTEMANN, P., and FOLIGUET, J. M. BLOCK, 1981. Disinfection des eaux par I'ozone. Eau Ind. 58: 69-76. K., and HERBOLD, K. 1988. Abotung von Hepatitis A BOTZENHART, Virus im Wasser durch Ozon. Z. Gesamte Hyg. 34: 508-5 10. G. R., MURRAY, T. M., and POLLARD, M. 1975. BURLESON, Inactivation of viruses and bacteria by ozone, with and without sonication. Appl. Microbiol 29: 340-344.

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Effects of ozone treatment on the infectivity of hepatitis A virus.

The inactivation of a large-focus-forming variant of hepatitis A virus (HM-175) by ozone was investigated. Experiments using mainly single-particle vi...
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