Processes Controlling Virus Inactivation in Seawater Ralph Mitchell Division of Engineering and Applied Physics, Harvard University, Cambridge, Mass. 02138
H. W. Jannasch Woods Hole Oceanographic Insiitution, Woods Hole, Mass.02543
The rate and extent of inactivation of bacteriophage ax174 in natural seawater was found to be controlled simultaneously by biological and abiological factors. A specific antagonistic group of microorganisms developed when the phage was added to seawater. In addition, a direct chemical inactivation was detected in filter-sterilized seawater. A protective effect for the survival of the phage was shown to be related to the presence of microbial cells killed either by autoclaving or by ultraviolet irradiation. In natural seawater, the combined action of biological and chemical antiviral activities is believed to be substantially stronger than the protective effect exerted by the detrital organic material.
T
he processes in seawater responsible for eradication of nonmarine microorganisms have been discussed in a recent review (Mitchell, 1968). The destruction of Escherichia coli in seawater has been associated with the activity of marine microorganisms capable of causing lysis of the enteric bacteria (Mitchell, Yankofsky, et al., 1967; Mitchell and Yankofsky, 1969). It is surprising that, despite the dangers of transmission of enteric diseases by viruses carried into seawater in sewage, comparatively little is known about the processes leading to the inactivation of viruses in the sea. Metcalf and Stiles (1967) immersed dialysis tubes containing enteric viruses in coastal waters and studied virus survival. They observed a rapid rate of inactivation and suggested that the abundant estuarine flora might be involved in the kill. In our current investigation, the authors are concerned with the contribution of the native marine microflora to the destruction of a virus added to fresh, natural estuarine water. Materials and Methods
The ax174 strain of bacteriophage used in this study was obtained from D. T. Denhardt, Biology Department, Harvard University. This virus is specific for Exherichin coli C, and has been described by Sinsheimer (1959) as a hexagonal, single-stranded DNA virus with a diameter of 30 mp. The host was E. coli C. Bacteriophage particles were counted by the double layer plate method described by Adams (1959). NaCl (OSZ) was added to the nutrient agar, and nutrient broth was used as the dilution medium. Seawater was sampled from the Eel Pond, Woods Hole, Mass., in July and August 1968. The bacterial count was determined using nutrient agar (Difco) prepared in natural seawater and measured lo6 to lo6 per ml. in the pond. Fresh samples of seawater were used at all times.
The seawater samples with added bacteriophage were incubated in 100-ml. quantities in 250-ml. Erlenmeyer flasks on a reciprocal shaker at 25' C. The virus was added at a concentration of approximately 10I2particles per ml. following washing and resuspension in sterile seawater. Marine microorganisms were concentrated by centrifugation at 3000 x G for 20 minutes in a Sorvall refrigerated centrifuge and resuspended in sterile seawater. Marine bacteria were counted on seawater nutrient agar following incubation at 22' C. for 5 days. Dilutions were made in sterile seawater. The seawater was sterilized either by autoclaving for 20 minutes; by filtration through 0.22-micron pore size Millipore filters; or by ultraviolet irradiation. The ultraviolet irradiation was carried out by placing 10 ml. quantities of seawater in petri dishes under a shortwave ultraviolet light (less than 300 mp) for 30 minutes. No growth was detected in samples plated on seawater nutrient agar following this treatment. Results
Microbiological Inactivation. To determine the antiviral activity of the marine microflora, bacteriophage 9x174 was added to samples of fresh natural seawater at a concentration of approximately 10I2 per ml., and incubated at 25' C. Survival of the virus was checked at daily intervals. The results shown in Figure 1 indicate that in natural seawater the virus was rapidly destroyed. The titer declined from 10I2per ml. to 103 per ml. in 6 days. Antiviral activity was diminished by autoclaving the seawater. The virus was relatively stable in autoclaved water for more than two weeks. These results suggested that a portion of the native marine microflora was involved in the decline of bacteriophage ax174 in the seawater. Further evidence of this antiviral activity of the normal flora was obtained in experiments with autoclaved seawater to which known quantities of marine microorganisms had been added. The microorganisms were collected by centrifugation of fresh seawater and returned to the sterile seawater at different concentrations. The quantities were determined by colony counts on seawater nutrient agar. The data in Figure 2 show that the rate of decline of the virus in seawater was proportional to the concentration of the marine microflora. Addition of l o 3 marine bacteria per ml. of seawater in this manner resulted in a decline in the count of virus from 1012 per ml. to 108 per ml. in 6 days. When lo6 bacteria per ml. were added to the seawater the virus count declined from 1012 per ml. 105 per ml. in the same period of time. The quantitative response of the marine microflora during the decline of added bacteriophage was determined and the resulting data are shown in Table I. In the absence of the virus, no appreciable change in the concentration of marine microorganisms was detected. These data indicate that an Volume 3, Number 10, October 1969 941
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Table I. Relationship Between the Concentration of the Marine Microflora and Rate of Decline of Bacteriophage *X174 in Seawater No. Marine Bacteria No. Virus Particles Time, per MI. Seawater per MI. Seawater days 0 4.0 X los 10'2 5 5 . 5 x 106 107 8 4 . 0 X lo6 108 10 2 . 0 x 107 10s
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Figure 1. A comparison of the rate of inactivation of bacteriophage ax174 in autoclaved, UV-irradiated, and fresh natural seawater incubated at 25" C.
antagonistic marine microflora develops following the addition of a virus to seawater. Chemical Inactivation. A direct effect of chemical components of seawater on the virus was detected in filter-sterilized seawater. The results are shown in Figure 3. Seawater filtered through 0.45-micron filters also displayed antiviral activity per ml. to loa per ml. in 6 reducing the virus titer from days. Bacterial counts showed that filtration through 0.45micron filters did not remove all of the microorganisms, whereas seawater filtered through 0.22-micron filters appeared to be sterile. The water displayed an even stronger activity after passage through a 0.22-micron filter. No virus could be detected following 6 days of incubation (Figure 3). Apparently, in addition to a microbiological destruction of the virus in seawater, a chemical fraction of the water is associated with the inactivation. This chemical inactivation may be connected either with the relatively high concentration of heavy metals or with the salinity of the seawater. Protective Effect of Suspended Organic Material. The results obtained with seawater sterilized by filtration instead of autoclaving indicated that a third factor was involved in the destruction of viruses. The results ;n Figure 3 show that anti-
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Figure 2. The effect of the size of the marine microflora on the inactivation of bacteriophage ax174 in seawater 942 Environmental Science & Technology
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Figure 3. Inactivation of bacteriophage ax174 by chemical components of seawater
viral activity increased when the natural microflora was removed. A study was undertaken to determine a possible effect of the organic material represented by the suspended cells and the relationship of such an effect to the biological and chemical inactivation of the virus. The microbiological antiviral activity was removed by autoclaving or UV-irradiation. Two different sterilization techniques were used to detect a possible effect on the chemical antiviral activity. The data in Figure 1 show a nonspecific protective action probably exerted by the suspended dead cell material. This action was sufficient to prevent inactivation of the virus by chemical components of the seawater. The protection was eliminated when the suspended organic matter was removed by filtration through a 0.22-micron Millipore filter (Figure 3). This observation supports the suggestion that suspended organic matter protects the virus against inactivation in the seawater. Discussion
We have demonstrated in this study that survival of the bacteriophage *X174 is affected in natural seawater by three different processes. The observation that sterilization of the water reverses the antiviral activity indicates that a biological antagonistic factor is involved. Additional evidence was obtained from the observation that the rate and extent of kill of the bacteriophage was directly proportional to the number of marine microorganisms in the water. Metcalf and Stiles (1967) found that the decline of enteric viruses in estuarine waters displayed seasonal variations, and that survival times were prolonged when the seawater was polluted. They suggested a correlation between virus inactivation and the activities of the native flora of the estuarine water. In a study of the decline of Escherichia coli in seawater, an obligately parasitic bacterium, Bdellovibrio, is partially responsible for the kill of E. coli (Mitchell, Yankofsky, et al., 1967). In a later study the authors implicated a marine amoeba in the decline of E . coli from seawater (Mitchell and Yankofsky, 1969). Both of these organisms use bacteria as an essential substrate, and neither organism can be detected in conventional inert media. An analogous parasitic population may develop in response to the addition of a virus to seawater. Alternatively, a specific antagonistic bacterial community may develop, replacing the natural microflora, without an overall increase in the total population.
The increased antiviral activity of seawater observed when the native microflora was removed suggests that certain chemical components of seawater are capable of rapidly inactivating this virus. Pramer, Carlucci et a/., (1963) described a similar inactivation of enteric bacteria in seawater and attributed the effect to heavy metals. Microbial cells killed by UV-irradiation or by autoclaving appear to protect the virus against inactivation. Cookson and North (1967) have demonstrated adsorption of bacteriophage onto activated carbon and have pointed out that the virus is not inactivated by adsorption. Similarly, in seawater, adsorption onto organic particles may serve to protect the virus. In this study, three processes have been found to affect the survival of bacteriophage ax174 in seawater: inactivation by microorganisms; inactivation by chemical components of the seawater; and the protective action of organic particulate material. In natural seawater, the combined chemical and microbiological antiviral action appears sufficient to overcome the protective action of suspended organic matter, and the virus is rapidly inactivated. The nature of the chemical and microbiological agents causing inactivation of the virus are currently being investigated. Literature Cited Adams. M. R.. “BacterioDhaaes.” D. 450. Interscience. New - , York, 1959. ‘ Cookson. SCI.TECHNOL. 1. ~~. ~.~ J. T.. North. W. J.. ENVIRON. 46-52 (1967). ’ Metcalf, T.G., Stiles, W. C. in “Transmission of Viruses by the Water Route.” G. Berg. -. Ed., -DD. - 439-44, Interscience, N. Y., 1967. Mitchell, R., Water Research 2, 535-44 (1968). Mitchell, R., Yankofsky, S.,ENVIRON. SCI.TECHNOL. 3,5746 (1969). Mitchell, R., Yankofsky, S., Jannasch, H. W. Nature 215, 891-3 (1967). Pramer, D . ,Carlucci, A. F., Scarpino, P. V., in “Marine Microbiology,” pp. 567-72, C. H. Oppenheimer, Ed., Thomas, Springfield, Ill., 1963. Sinsheimer, R. L.,J . Molec. Biof.1 , 3 7 4 1 (1959). Received for review November 8, 1968. Accepted May 22, 1969. This work was supported in part by grant No. WP-00967 to Harvard University from the U. S . Department of The Interior, Federal Water Pollution Control Administration, and by Grant number GB-7747 of the National Science Foundation to the Woods Hole Oceanographic Institution (contribution number 2241).
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