ENTERIC VIRUSES IN GROUND AND SURFACE WATERS: A REVIEW OF THEIR OCCURRENCE AND SURVIVAL Elmer W. Akin, William H. Benton, and William F. Hill, Jr. ENVIRONMENTAL PROTECTION AGENCY Water Quality Office Division of Water Hygiene Gulf Coast Water Hygiene Laboratory Dauphin Island, Alabama 36528 1971 ------- Copyright © 1971. Reprinted by permission of the Board of Trustees of the University of Illinois from PROCEEDINGS THIRTEENTH WATER QUALITY CONFERENCE February 1971 VIRUS AND WATER QUALITY: OCCURRENCE AND CONTROL Edited by Vernon L. Snoeyink Department of Civil Engineering University of Illinois Environmental Protection Agency of the State of Illinois ------- ENTERIC VIRUSES IN GROUND AND SURFACE WATERS: A REVIEW OF THEIR OCCURRENCE AND SURVIVAL* ELMER W. AKIN, WILLIAM H. BENTON, AND WILLIAM F. HILL, JR. Environmental Protection AgencyWater Quality Officei division of Water Hygiene, Gulf Coast Water Hygiene Laboratory} Dauphin Island, Alabama 36528. ABSTRACT Enteric viruses have been isolated from surface water samples collected throughout the world. Investigations reviewed in this paper indicate that enteric viruses were isolated from an average of 36% of the surface water samples examined. These viruses are shed in the feces of infected man and animals-and may enter water systems by way of soil runoff and sewage, both treated and untreated. The increased frequency of wa£er reuse for domestic purposes has increased the probability of human contact vrith contaminated surface waters. Laboratory studies indicate that viruses tend to adsorb to soil parti- cles and would therefore be removed before they reach ground water or soon after entering the underground system. However, epidemiological studies have implicated both contaminated surface and ground water as the transmission route in a limited number of infectious hepatitis outbreaks. Remarkably, no w:despread waterborne epidemics of viral disease other than infectious hepatitis have been substantiated. The amount of endemic disease caused by contact with virus-contaminated surface waters is completely unknown. The transmission route of endemic viral disease is difficult to determine due to the large per- centage of asymptomatic infections which occur with these viruses. Numerous workers have studied the survival of enteric viruses in waters and have found them to survive for a significant length of time to con- sider water a potential route of viral disease transmission. They have shown that 2 - 100 days are required for various members of the enteric virus family to lose 99.9% of their initial infectivity when suspended in different surface waters at 20-25°C. Our flow-through experimental system has allowed us to study virus survival in estuarine water under quasi-natural conditions. In these experiments, poliovirus 1 infec- tivity was reduced by 99% in 4 hours at 30°C (summer conditions) and 5-15 hours at 22°C (autumn conditions). By yielding survival data indicative of that occurring in the" natural setting, field studies of this type may have a very practical application. With this information and viral isolation data, water treatment requirements and recycling- frequency guidelines may be realistically determined. *Portions of this work will be submitted by the senior author in partial fulfillment of the requirements for the degree of Doctor of Public Health, University of Michigan, Ann Arbor, Michigan. ------- INTRODUCTION Trask et al. (1938) reported in the late thirties their finding of the regular presence of poliovirus in the stools of poliomyelitis patients. A few years later workers sought _and detected human enteric viruses in domestic sewage. This occurred just as the field of virology was coming into its own. The then new in vitro cell-culture virus systems yielded an avalanche of virus data including the isolation and identification of viruses not yet associated with disease. The isolation of viruses excreted through the gut of man has increased to approximately 100 serotypes today, and more are sure to be discovered. Increasingly, the enteric viruses are being associated with diseases not even suspected of having a virus etiology a few years ago; i.e., diabetes mellitus (Gamble et al., 1969) and mongolism (Stoller and Collmann, 1965). These are in addi- tion to a long list of conditions known for some years to be caused by enteric viruses; i.e., meningitis, myocarditis, pericar- ditis, hepatitis, paralysis, rashes, febrile illness, and gastro- enteritis. The human health hazard of adenoviruses found to be tumorigenic for experimental animals remains to be adequately assessed. Since the disposal of human waste inevitably involves water, and since the recycling time for domestic and industrial water usage has continually diminished with population and industrial growth, and since every person regardless of age consumes some water ------- -2 each day of his life, it is imperative that we know the fate of infectious and dangerous substances that enter our water systems. Enteric viruses can be readily isolated from domestic sewage and present treatment methods do not produce virus-free effluents (Kollins, 1966; Grabow, 1968). In addition, sewage from approxi- mately 60% of the population of the United States receives no significant treatment before it enters a disposal area, usually a body of water (Holcomb, 1970). Human enteric viruses have also been isolated from rectal samples of domestic and wild animals, identifying soil runoff as another potential source of water contami- nation (Kalter, 1967; Lundgren et a!., 1968; Grew et al., 1970). With this empirical knowledge, we can deduce that human enteric viruses are present to some degree in most natural waters. GROUND WATERS The possibility of ground water viral pollution has received very little concern. However, laboratory studies have shown that viruses readily adsorb to a wide variety of substances including natural clays and silts. Carlson et al. (1968) have reported the effective adsorption of bacteriophage T2 and poliovirus to kaolinite 4, montmorillonite 23, and illite 35 in the presence of electrolytes. Jakubowski (1969) confirmed these results with poliovirus 1 and extended his observations with clays and marine silt. This adsorp- tion was found to be reversible. Free infectious virus could be eluted from the particles in the presence of a proteinaceous ------- -3 solution. Drewry and Eliassen (1968) have found bacteriophages Tl, T2, and fZ to effectively adsorb to nine different soil types taken from California and Arkansas. Most workers emphasize the influence of pH and cation concentration on virus adsorption. Increasing adsorption occurs with the reduction of pH below 8.0 and with the addition of cations, especially the divalent species. Romero (1970) has recently reviewed much of the literature on the movement of bacteria and viruses through porous media. He reported that soil uniformly composed of fine sand with a high clay content is the best aquifer material for removing biological contaminants and concludes that these pollutants are able to travel a maximum of 100 feet in this environment. Robeck et al. (1962) found that two feet of packed sand removed poliovirus 1 from slow moving water but penetration of near 100% of the virus occurred as the flow rate was increased. Hori et al. (1970) recently reported that poliovirus 2 was able to penetrate a 6-inch column of three types of Oahu (Hawaii) soil rather rapidly, even though a significant reduction in virus titer was found in the filtrate. Considering these findings, one would expect ground water to be cleansed of viruses by entrapment and adsorption after traveling a short distance. Bound viruses would thereby be broken down by soil microorganisms and environmental conditions as is other organic matter. However, sensitive methods are lacking to readily detect viruses at the levels they would probably occur in ground water. ------- -4 Therefore, confirmation or rejection of laboratory findings as they apply to the natural setting must be sought by other methods. Indeed, epidemiological findings have helped in this regard. Taylor et at. (1966) have reviewed the data oil 48 outbreaks of suspected waterborne infectious hepatitis involving approximately 31,000 people. He found well or spring water to be the suspected source of the virus iti 21 of the 48 outbreaks. Perhaps the early work of Neefe and Stokes (1945) provides the best quantitative data- They convincingly showed that a covered well providing drinking water at a summer camp was contaminated by the infectious hepatitis agent(s) fro® a cesspool 75 feet away. Tucker et al. (1954) similarly found that a spring supplying water to a campground was contaminated by faulty sever lines "near" the spring. Shuval (1970) has recently isolated enteric viruses from, a municipal well which was under surveillance as a possible common source for localized infectious hepatitis cases. These data would indicate that some enteric viruses are able to travel a finite distance through soil and enter a water supply. Of course, channels and breaks in subsurface rock formations may have been followed by the contaminated water rather than an actual soil percolation, thus allowing the viruses to travel much farther than laboratory data would indicate. Epidemiological findings seem to be at odds with laboratory data concerning the cleansing of ground water by filtration and ------- -5 adsorption. However, it must be kept in mind that the epidemiological findings concern the infectious hepatitis virus only and the laboratory data were obtained with lab-strains of poliovirus and bacteriophage. In addition, the data are so limited and the factors so variable in the natural settings that it remains impossible to draw general conclusions as to the health hazard associated with the utilization of ground water near a pollution source. SURFACE WATER Considerable data are available concerning the viral contamination of surface waters. Using recovery methods realized to be quantita- tively questionable, field surveys have shown human enteric viruses to be present in rivers and estuaries around the world. Coin et at. (1964) using a swab method found 21% of 1156 samples of river water around Paris to be positive for enteric viruses. Foliquet et at. (1966) found viruses in river and drinking water (9 and 8% of samples positive, respectively) from French communities along the Meurthe and Moselle Rivers. Bagdasar'yan (1968) reported 34% of 164 river water samples collected within the city limits of Moscow contained enteric viruses. Farrohi (1966) observed that 38 - 63% of his samples from two Swiss rivers emptying into Lake Geneva contained enteric viruses. Shuval (1970) isolated enteric viruses from 3 of 34 Jordan River samples collected up to 15.5 miles from the primary contamination source. The Metropolitan Water Board of London has determined that 56£ of 15 intake water samples (Thames River) were ------- -6 positive for enteric viruses (Taylor, 1969). In this country, Metcalf and Stiles (1968) have found 27 - 52% of their water samples taken from polluted tidal rivers contained enteric viruses. Twenty- seven percent of the water samples from the receiving bay were virus positive, Lamb et at, (1964) isolated enteric viruses from 27% of 71 upper-Illinois River samples one mile from a sewage outfall. Grinstein et at. (1970) have used a newly developed concentration method for sampling river water in the Houston (Texas) area. Nearly 100% of the samples collected 5 miles downstream from the nearest sewage outfall were positive for enteric viruses. These selected reports unquestionably document the fact that enteric viruses shed from their natural host by the fecal route survive for a significant length of time in an aqueous environment to become potential health hazards. Laboratory studies have been conducted which were aimed at elucidating major factors affecting virus survival, Prier and Riley (1967) have attempted to single out some of the factors exhibiting an effect on virus survival in water (Table 1). Of the factors listed, temperature is the only one which is well defined and consistent. Under otherwise identical conditions, virus survival is inversely related to temperature. This relationship is seemingly true throughout the entire temperature range. Figure 1 exemplifies the effect of temperature upon the inactivation curve of two poxviruses. Note the two types of curves observed; i.e., ------- TABLE 1 FACTORS THAT MAY AFFECT VIRUS SURVIVAL IN WATER (PRIER AND RILEY, 1967) 1. Time virus is in water \ 2. Nature of water (lake, stream, well) 3. Rate of water flow 4. Temperatures of water 5. Chemical content of water 6. Organic content of water ------- o 40 2 \ K 2 50 45 55 20 40 60 MINUTES 50 52.5 60' 20 40 60 MINUTES FIGURE 1. Thermal inactivation in aqueous environments of (A) variola virus (Hahon and Kozikowski, 1961) and (B) vaccinia virus (Kaplan, 1958). ------- -7 one component curves with variola virus (A) and two component curves with vaccinia virus (B). Factors other than temperature have been less precisely defined. Purity of water seems to have a significant effect on survival time. In general, enteric viruses have been found to survive longer in distilled water than in polluted water and interestingly, longer in "grossly" sewage-polluted water than in "moderately" polluted water (Table 2). Cioglia and Loddo (1962) reported that the addition of sewage to river and seawater increased the survival time of selected enteric viruses. It has been suggested that gross pollution provides surface water an ample supply of proteinaceous material which protects the virus and reduces available oxygen thereby retarding oxidation of the virion. These processes may be Involved but do not explain the shortest survival time in the moderately polluted water. A number of workers have studed the survival of enteric viruses under laboratory and field conditions. Table 3 Is an attempt to present some of these findings in a form permitting as much comparison as possible. Data are arranged from the shortest to the longest survival time found to be required for a 3 log (99.9%) inactivation at 20 - 25°C without regard to the virus type. Much pertinent information is of necessity omitted from the table; e.g., method of titration, characterization of suspending waters, experimental conditions. Nevertheless, a general pattern of ------- TABLE 2 AVERAGE TIME IN DAYS OF 99.9% REDUCTION IN TITER OF INDICATED ENTERIC VIRUSES WHEN SUSPENDED IN WATER WITH THREE LEVELS OF DOMESTIC POLLUTION AT 3 TEMPERATURES (CLARKE, et at., 1964) Amount of pollution Phldovirus 1 Echovirus 7 Echovirus 12 Coxsackievirus A-9 2B°C 20°C 4°C 28°C 20°C 4"C 28°C 2Q°C 4eC 28°C 20°C 4°C Low (river water) Moderate (river water) Heavy (sewage) 17 20 27 11 13 19 17 23 110 12 16 26 5 7 15 28 41 130 5 12 33 3 5 19 20 32 60 <8 <8 10 5 8 20 6 N.D* 12 * N .D. - Hot done. ------- TABLE 3 SS OF INFECTIVITY OF ENTERIC VIRUSES WHEN SUSPENDED IN VARIOUS AQUEOUS ENVIRONMENTS 'pe of water References Virus Temperature (°C) 4-6 15-16 20-25 ja or ftuarine water Cioglia & Loddo (1962) Shuval (1970) Akin, et at. (UP)* Matussian & Garabedian, (1967) Lycke, et at. (1965) V Akin, et at, (UP) Akin, et at, (UP) Akin, et at. (UP) McLean & Brown (1968) Shuval, et at. (1971) Cioglia & Loddo (1962) Cioglia & Loddo (1962) Cioglia & Loddo (1962) Cioglia & Loddo (1962) Metcalf & Stiles (1967) Metcalf & Stiles (1967) Metcalf & Stiles (1967) Coxsackie B-3 Polio 1 Coxsackie B-l Polio 1 Polio 3 Reo 1 Echo 6 Polio 1 Polio 2 Polio 1 Polio 1 Polio 2 Polio 3 Echo 6 Coxsackie B-3 Echo 6 Polio 1 Daye/tog titer reduction 90/3 9/2 45/3 60/3 30/3 30/3 88/3 130/3 8/3 9/3 15/3 15/3 8/3 15/3 14/3 16/3 15/3 2/3 2-6/3 3/3 3-6/3 4/3 4/3 4/3 5/3 5/3 8/3 8/3 8/3 8/3 15/3 28/3 fcble 3 continued. ------- Page 2 of Table 3 BLE 3. LOSS OF INFECTIVIXY OF ENTERIC VIRUSES WHEN SUSPENDED IN VARIOUS AQUEOUS ENVIRONMENTS - CONT'D. ype of water References Virus Temperature (°C) 4-6 15-16 20-25 «Jal river water Akin, et al. (UP) Akin, et al. (UP) Akin, et al. (UP) Akin, et al. (UP) Coxsackie B-l Reo 1 Echo 6 Polio 1 Days/log titer reduction 3/3 1 5/3 6/3 6/3 Cioglia & Loddo (1962) Coxsackie B-3 75/3 8/3 2/3 Prier & Riley (1967) Echo 6 7/0.5 - 3/3 Prier & Riley (1967) Polio 1 7/1 - 3/3 Prier & Riley (1967) Coxsackie B-3 7/1.7 - 3/3 Clarke, et al. (1964) Echo 12 19/3 - 5/3 Clarke, et al. (1964) Echo 7 15/3 - 7/3 Clarke, et al. (1964) Coxsackie A-9 10/3 - <8/3 Cioglia & loddo (1962) Polio 2 75/3 15/3 8/3 Cioglia & Loddo (1962) Polio 3 30/3 8/3 8/3 Cioglia & Loddo (1962) Echo 6 60/3 15/3 8/3 Clarke, et al. (1964) Coxsackie A-9 20/3 - 8/3 Clarke, et al. (1964) Echo 12 33/3 - 12/3 Clarke, et al. (1964) Polio 1 6/3 - 13/3 Cioglia & Loddo (1962) Polio 1 60/3 45/3 15/3 Clarke, et al. (1964) Echo 7 26/3 - 16/3 table 3 continued ------- Page 3 of Table 3 fe 3. LOSS OF INFECTIVITY OF ENTERIC VIRUSES WHEN SUSPENDED IN VARIOUS AQUEOUS ENVIRONMENTS - CONT'D. |>e of water Temperature (eC) References Virus 4-6 15-16 20-25 Day8/log titer reduction Clarke, et al. (196A) Polio 1 27/3 - 20/3 Poynter (1968) Polio 3 50/3 18/3 - Taylor (1967) Polio 3 67/3 7/1.3 7/2 Clarke & Chang (1959) Coxsackie A-2 - - 5/2 Clarke & Chang (1959) Coxsackie A-2 - - 47/2 Taylor (1967) Coxsackie B-5 - 24/1 - Poynter (1968) Coxsackie B?** 18/2 - - Prler & Riley (1967) Coxsackie B-3 7/1.7 - 3/3 Prier & Riley (1967) Polio 1 7/1.5 - 3/3 Clarke, N. A. (UP) Echo 7 22/3 - 4/3 Prier & Riley (1967) Echo 6 5/3 - 5/3 Clarke, N. A. (UP) Coxsackie A-9 6/3 - <6/3 Clarke, N. A. (UP) Polio 1 27/3 - 6/3 Joyce & Weiser (1967) Coxsackie ? 18/3 - 6/3 Clarke, N. A. (UP) Echo 12 14/3 ¦m* <6/3 Joyce & Weiser (1967) Polio ? 21/3 - 10/3 Joyce & Weiser (1967) Echo ? 23/3 - 12/3 Joyce & Weiser (1967) Echo ? 21/3 - 20/3 ir water it'd.) t unded fresh ater 3 continued ------- rage 4 of Table 3 E 3. LOSS OF INFECTIVITY OF ENTERIC VIRUSES WHEN SUSPENDED IN VARIOUS AQUEOUS ENVIRONMENTS - CONT'D. te of water References Virus Temperature (°C) 4-6 15-16 20-25 funded fresh *r - Cont'd. Joyce & Welser (1967) Polio ? Joyce & Weiser (1967) Polio ? Joyce & Weiser (1967) Echo ? Day $/log titer reduction 52/3 52/3 42/3 21/3 22/3 24/3 Clarke, N. A. (UP) Echo 7 85/3 10/3 Clarke, N. A. (UP) Echo 12 130/3 11/3 Clarke, N. A. (UP) Coxsackie A-9 98/3 15/3 Clarke, N. A. (UP) Polio 1 140/3 95/3 McLean & Brown (1968) Polio 2 12/No loss 5/1 Clarke & Chang (1959) Coxsackie A-2 <•* « 100/2 Taylor, (1967) Polio 3 167/3 - Poynter, (1968) Polio 3 168/3 - onized or tilled water Akin, et al. (UP) Akin, et al. (UP) Akin, et al. (UP) Akin, et at. (UP) Poynter, (1968) Poynter, (1968) Reo 1 Coxsackie B-l Echo 6 Polio 1 Coxsackie B? Polio 3 95/2 180/1 3/3 5/3 14/3 11/1 56/1 Unpublished data Virus serotype not reported ------- -8 inactivation as well as variability within and between enteric virus survival times may be gleaned from the table. ESTUARINE FIELD STUDIES Most of the survival data herein reported were obtained under laboratory or artificial conditions which may not yield results representative of the natural setting. This fact has concerned us at the Gulf Coast Water Hygiene Laboratory and we have therefore initiated survival studies under more natural conditions. A diagrammatic representation of our experimental system is shown in Figure 2. "Fresh" estuarine water was continuously pumped from Little Dauphin Island Bay into a tank in our wet laboratory with the overflow being returned to the bay. Water for these experiments was siphoned from the wet laboratory to the constant head tank located just outside the laboratory. From there, the estuarine water was siphon-fed into the 1000-gallon fiber glass tank at a rate of 7 liters/min, Water passing through the distribution manifold was mixed with the 1000 gallons of estuarine water in the tank by electric stirrers. Excess water then flowed out the central standpipe and thence through a flow meter. In this flow-through system, marine life and biological metabolites are not allowed to accumulate to a level alien to that found In the natural setting. Therefore, this system should yield virus survival patterns more indicative of those which actually occur in the complex estuarine environment. Temperature was recorded constantly. Salinity, ------- CONSTANT-HEAD TANK FLOW TEMPERATURE RECORDING PROBE ELECTRIC STIRRER OVERFLOW 2M0' DISTRIBUTION MANIFOLD 1,000 GALLON FIBERGLAS TANK FLOWMETER FIGURE 2, Flow-through system for studying the survival of virus in estuarine water. ------- -9 turbidity, and flow rate measurements were made Intermittently throughout the experimental period. The fiber glass tank was filled with estuarine water from the wet laboratory on the day before an experiment was run. A Plexiglas cylinder 5 cm x 94 cm was inserted vertically into the tank and filled with distilled water (approxi- mately 1700 ml). Since laboratory data have previously been collected on the inactivation curve of poliovirus suspended in distilled water at 23°C, this Plexiglas unit when dosed with virus provided a virus control and a comparison for the inactivation patterns obtained in the flow-through system. On the morning of the experiment a predetermined multiplicity of poliovirus 1 was added to the tank and cylinder. After 30 minutes of mixing, the initial samples were taken and the incoming siphon begun. At each sampling interval, a pool of 100 ml was obtained by collecting 10 ml from 10 locations within the tank. From this pool, 5 ml were removed in duplicate and placed into a test tube containing lyophilized nutrient broth which provided protective proteins for the virus without significantly diluting the sample. Ten-ml portions were also removed from the distilled water cylinder and handled in the same manner as the tank samples. These samples were immediately placed at -80°C and held at that temperature until assayed for virus by the plaque technique using HEp-2 cell monolayers. Three ml of each sample were assayed with results reported as the mean PFU/ml of sample. ------- -.10 The reduction in plaque counts at each sampling interval represented a virus loss due to dilution as well as inactivation. To differentiate loss by dilution from loss by inactivation, a correction factor (CF) was obtained in the following manner. Either immediately prior to or following each experiment, the 1000-gallon tank was filled with high salinity estuarine water and the salinity determined hydrometrically. The mixers were engaged and tap water having a negligible salinity (0.2 - 0.4 ppt) was introduced into the tank through the distribution manifold. Hourly salinity determinations were made from samples collected at the tank effluent. Flow rate determinations were made intermittently throughout the sampling period. The reduction in salinity occurred geometrically and proceeded as a linear first order react* ->n. Therefore, the rate of salinity reduction (dilution) could be — (Qrhbt) calculated from the equation, N^/N = e , where is the initial salinity at time zero; is the salinity at time t\ a is the intercept and b is the reduction rate (slope function). A curve can be drawn of the form Y - a + bx which produces a straight line of best fit to the points observed. Since the kinetics of salinity reduction in this system should be practically identical to virus dilution in the experimental system, the slope of the line should be the same in both cases. Under the conditions employed, the Initial tank contents were diluted in half every 7-8 hours. The correction factor for each time interval during an experiment was ------- -11 calculated from the slope function and intercept of this line. The actual plaque count at each time interval was multiplied by the appropriate correction factor to obtain the calculated plaque count If no dilution had occurred. Four experiments have been conducted to date; two under summer- conditions with a temperature range of 28 - 33°C [salinity, 21 - 27 ppt; turbidity, 28 - 36 Jackson Turbidity Units (JTU)] and two under autum conditions with a temperature range of 20 - 24°C (salinity, 18 - 21 ppt; turbidity, 32 - 44 JTU). The results of these four experiments are shown in Figure 3. The two summer experiments are represented by a single inactivation curve since the calculated least squares curves were practically identical. As can be seen, lose in poliovirus infectivity in the summer experiments occurred exponentially with a 4 log (99.99%) reduction occurring in seven hours. The inactivation curve of the autumn experiments, however, began to "tail" between 4 and 8 hours resulting in a 2.5 and 3.5 log reduction in 24 hours for autumn experiments one and two, respectively. Even though temperature, salinity, turbidity, and flow rate measure- ments were very similar for the two autumn experiments, a one log difference in virus reduction occurred in the 24-hour samples. The loss of virus Infectivity in the distilled water cylinders did not exceed 0.5 log in six hours under summer conditions and did not exceed 0.5 log in 24 hours under autumn conditions* ------- 4 8 12 16 20 24 HOURS FIGURE 3, Inactivation of poliovirus 1 in estuarine water under field conditions during summer (0, A) and autumn (0* A) months. ------- -12 DISCUSSION AND CONCLUSIONS The exact method of viral inactivation in nature is not clearly understood. Pohjanpelto (1962) and Lund (1963) have reported that oxidation is important in the Inactivation of poliovirus above 50°C but plays a very small role in spontaneous inactivation at temperatures below 37°C, It still remains obscure as to which part of the virion participates in the oxidative inactivation at the higher temperature and the ill-defined inactiva- tion process at lower temperatures. Dinnaock (1967) has suggested that loss' of iufectivity can occur by nucleic acid or protein damage and that the stability of each moiety varies with temperature. Inactivation at a given temperature would therefore take place through whichever component is the least stable at that temperature. Regardless of the method of inactivation, the reviewed data clearly show that iriactivation time is inversely related to temperature and that a matter of days to weeks are required for a 99,9% inactivation of various enteric virus types at prevailing surface water temperatures. The wide time-range required for a 3 log (99.9%) reduction indicates that significantly different inactivation rates occur within the enteric virus family and that major factors other than temperature are important in inactivation (or survival) of these particles in the aqueous environment* These other factors have not been clearly defined. ------- -13 Enteric viruses shed from the gut of animal hosts are able nevertheless to survive for a significant length of time in natural bodies of water to spread disease by this route. Of the reports reviewed, an average of 36% of the water samples collected contained one or more viruses. In addition, this transmission route has been' well documented in a limited number of explosive outbreaks of infectious hepatitis. The most recent outbreak involved 3.8% of the population of a French town with evidence pointing to an extensively contaminated municipal water distribution system as the culprit (Gavan and Nutt, 1970). These outbreaks are rare and it seems safe to say that widespread viral morbidity and mortality are not occurring in this country as a result of contact with surface waters. The extent of endemic disease spread by enteric-virus- contaminated water, however, is anyone's guess. Epidemiological studies designed to answer this question are extremely costly and to date have not obtained sufficient support to initiate adequate investigations. Many enteric viruses produce an asymptomatic response in 90% or more of the persons infected, thereby producing a confusing transmission pattern when overt disease cases alone are considered. Costly and time consuming antibody studies conducted on a latfge segment of the population are required to determine infectivity patterns and therefore an accurate transmission picture. Less costly viral survival studies yield data that can ------- -14 provide an indirect evaluation of the health hazard.of human contact with water known to have received viral contaminated materials. It should be emphasized that our present water supply is being recycled for domestic and Industrial usages at an increasing frequency. This, of course, allows less time for nature's purifi- cation process to accomplish its task. Knowledge of viral survival patterns in natural water systems could be put into immediate practical use. These data coupled with the results of virus monitoring (assuming that a satisfactory concentration- isolation procedure is forthcoming) would provide a sound basis for determining water treatment needs and safe reuse frequency. The fate of enteric viruses in estuarine as well as fresh water has health importance. The increased coastal population growth has been accompanied by Increased exposure of recreational and seafood-producing coastal waters to domestic waste. Several workers have studied virus survival in seawater and have found It to possess a virucidal property (Magnusson et at., 1966; Matossian and Garabedian 1967; Shuval et al.t (1971). The work of Magnusson et al., (1967) and Shuval et al., (1971) indicates that a viable marine bacterium is responsible and that filtration or/heating the seawater to 90°C removes the virucidal property for poliovlrus. Conversely, Matossian and Garabedian (1967) using poliovlrus 1 found filtration or boiling had little or no effect upon the ------- -15 virucidal properties of Mediterranean Sea samples. Likewise, our studies show that filtration and autoclaving does not eliminate the virucidal property of Gulf Coast estuarine water upon poliovirus 1 (unpublished data). Mitchell and Jannasch (1969) studying 3>X 174 bacteriophage have concluded that three major factors are working in seawater to influence phage survival: (1) inactivating micro- organisms, (2) inactivating chemicals, and (3) protective organic matter. Considering the number of possibilities within each of the three factors, there can be an almost infinite number of combinations exerting an effect on virus survival. It may be practically impossible to isolate each combination of factors which significantly lengthens survival time or hastens inactivation of a virus particle in the various aqueous environments. Even if the combination of factors were elucidated, the result of their interactions may be so complex that virus survival predictions without actual field testing would be ill-advised. For the above reasons, we have begun conducting our survival studies under conditions more representative of nature but yet controlled to the extent that results lend themselves to interpre- tation. From the four experiments we have conducted in our flow- through system, the effect of temperature is clearly seen. A temperature reduction of a mean of 9°C from 31°C extended the 99.9% inactivation time three-fold (to a mean of 18 hours). It is interesting to note that the. inactivation curve of the two summer ------- experiments, conducted one month apart, are practically identical. The two autumn experiments, conducted 3 weeks apart, gave similar inactivation patterns but at a significantly different order of magnitude; i.e., approximately one log difference after 24 hours (Figure 3). This difference may reflect the occurrence of virucidal biological shifts in the marine environment during the change in seasons which were not apparent from the monitored environmental parameters. Also of interest is the change in inactivation rate occurring in the fall experiments between 4-8 hours. We may speculate that the initial rapid drop in virus titer does not represent inactivation at all but rather aggregation of virus particles to each other and to "marine silt." This could occur by the adsorption of virions in the presence of divalent cations in the seawater. Therefore, the flatter slope may represent the inactivatioii of stable configurations of virus aggregates. Chang (1967) has strongly emphasized the influence of clumping on viral survival curves. However, we do not rule out the possibility of the presence of multiple inactivation forces which may dominate during different phases of the inactivation period thereby changing the inactivation rate. Laboratory studies are being planned to clarify the aggregation possibility. Field studies Under winter and spring conditions are also being planned in an effort to determine the seasonal variability in virus survival time as well as the survival patterns of poliovirus 1. ------- -17 We have found a significantly different inactivation rate of poliovirus 1 under laboratory conditions versus our flow-through system when fresh estuarine water was used in both cases. A reduction in infectivity of 99.9% occurred in 72 hours in the laboratory studies contrasted to a mean of 18 hours in the flow-through system- at 23°C (unpublished data). Likewise, Metcalf and Stiles (1967) found that poliovirus 1, echovirus 6, and coxsackievirus B-3 survived longer under laboratory conditions contrasted to suspension of these viruses in an estuary within cellulose dialysis tubes. This phenomenon still remains to be explained, and when all published findings are considered, major factors affecting the inactivation of enteric viruses in the natural marine environment are yet to be elucidated. If our flow-through experimental system is at all representa- tive of a "real" estuarine environment, then it would seem that at least one enteric virus (poliovirus 1) is inactivated at a faster rate in nature than in the laboratory setting. Assuming that this is true for all enteric viruses, then laboratory survival studies have a built-in margin of error on the side of safety. This would be welcome from a public health standpoint by allowing some added tolerance in making viral hazard predictions based on laboratory survival studies of highly pathogenic viruses. Of course, the elusive infectious hepatitis agent(s) still remains the villain a 8upeviov of water transmissible Infectious agents. The limited data ------- -18 available Indicate it may be many times more resistant than any known virus to nature's purification process as well as to chemical disinfectants. However, until it is isolated and adequately studied, viral hazard predictions based on survival studies must be obtained with the most resistant enteric virus readily available for study. This determination is yet to be made. ACKNOWLEDGMENTS We thank William Zirlott and Clinton Collier for their assistance in setting up and maintaining the flow-through experimental system. DISCLAIMER CLAUSE Mention of any commercial products in this paper does not imply endorsement by the Federal Government. ------- REFERENCES -19 Bagdasar'yan, G. A. (1968) "Investigation of Riverwater for Enteroviruses," Hygiene and Sanitationt 33:10-12, pp.134-135. Carlson, G. F., Jr., Woodard, F. E,, Wentworth, D. F., and Sproul, 0. J. (1968) "Virus Inactivation on Clay Particles in Natural Waters," Journal Water Pollution Control Federa- tiont 40:2, pp. R89-106. Chang, S. L, (1967) "Statistics of the Infective Units of Animal Viruses," in Transmission of Viruses by the Water Route, G. Berg, ed, New York: Interscience, Cioglia, L., and Loddo, B. (1962) "The Process of Self-Purification in Marine Environment. Ill, Resistance of Some Enteroviruses," Nuovi Annali Di IgieneE. lliorobiologiat 13,pp. 11-29. Clarke, N. A,, and Chang, S, L, (1959) "Enteric Viruses in Water," Journal American Water Works Association 51, pp. 1299-1317. Clarke, N, A,, Berg, G., Kabler, P. W., and Chang, S. L, (1964) "Human Enteric Viruses in Uater: Source, Survival, and Removability," in International Conference on Water Pollution Researcht London, 1962, New York: Pergamon. Coin, L., Menetrier, M. L., Lobonde, J,, and Hannoun M, C. (1964) "Modern Microbiological and Virological Aspects of Water Pollution" in Advances Water Pollution Research, Proceedings 2nd International Conference, 0, Jaag and J. K. Boors, eds, Oxford: Pergamon (1965), Drewry, W, A,, and Eliassen, R. (1968) "Virus Movement in Groundwater," Journal Water Pollution Control Federation, 40, pp. R257-271. Dimraock, N. J. (1967) "Difference Between the Thermal Inactivation of Picornaviruses at "High"and "Low" Temperatures," Virology, 31, pp. 338-353. Farrohi, K. (1966) "Research on Viruses in Water: Experimental Studies, Consequences of these Studies in Problems of General Hygiene," Revue Irmunolc^y Ther. Antimicrob, t 30:6, pp. 355-356. ------- -2° jl'oliquet, J. M., Schwartzbrod, L,, and Gaudin, 0. G, (1966). "La Pollution Virale des Eaux Usees, de Surface et D'alimentation," Bulletin World Health Organization, 35, pp. 737-749. ICamble, D. R., Kinsley, M. L», Fitzgerald, M. G,, Bolton, R., and Taylor, K. W. (1969) "Viral Antibodies in Diabetes Mellitus," British Medical Journal, 5671, pp. 627-630. Gavan, D. T., and Nutt, J. W. (1970) "An Epidemic of Waterborne Infectious Hepatitis in France," Aroh. Environ. Health, 20, pp. 523-532. Grabow, W. 0. K. (1968) "The Virology of Waste Water Treatment," Water Research, 2, pp. 675-701. Grew, N., Gohd, R. S., Arguedas, J., and Kato, J. I. (1970). "Enteroviruses in Rural Families and Their Domestic Animals," American Journal of Epidemiology, 91, pp. 518.-526. Grinstein, S., Helnick, J. L., and Wallis, C. (1970) "Virus Isolations from Sewage and from Streams Receiving Effluents of Sewage Treat- ment Plants," Bulletin World Health Organization, 42, pp. 291-296. Hahon, N., and Kozikowski, E. (1961) "Thermal Inactivation Studies with Variola Virus," Journal of Bacteriology, 81, pp. 609-613. Holcomb, R. W. (1970) "Waste-Water Treatment: The Tide is Turning," Science, 169, pp. 457-459. Hori, D. H., Burbank, N. C., Jr., Young, R. H. F., Lau, L. S., and Klemmer, H. W. (1970) "Migration of Poliovirus Type 2 in Perco- lating Water Through Selected Oahu Soils," Water Resources Research Center Tech, Report #Z6, University of Hawaii, Honolulu. Jakubowski, W., (1969) "Poliovirus Adsorption by Soil Particles in Seawater," Master of Science Thesis, Oregon State University. Joyce, G., and Weiser, H, H. (1967) "Survival of Enteroviruses and Bacteriophage in Farm Pond Water," Journal American Water Works Association, 59:4, pp. 491-501. Kalter, S. S. (1967) "Picornaviruses in Water" in Transmission of Viruses by the Water Route, G. Berg, ed. New York: Interscience. Kaplan, C. (1958) "The Heat Inactivation of Vaccinia' Virus" Journal General Microbiology, 18, pp, 58-63, Kollins, S. A. (1966) "The Presence of Human Enteric Virus in Sewage and Their Removal by Conventional Sewage Treatment Methods," In Advances in Applied Microbiology, 8, pp, 145-193. W. W. Umbreit ed. New York; Academic Press* ------- -21 Lamb, G. A., Chin, T. D. Y., and Scarce, L. E. (1964) "Isolations of Enteric Viruses from Sewage and River Water in a Metropoli- tan Area," American Journal Hygienej 80, pp. 320-327. Lund., E, (1963) "Oxidative Inactivation of Poliovirus at Different Temperatures," Arah. ges. Virusforscho, 13, pp. 375-386. Lundgren, D. L., Clapper, W. E., and Sanchez, A. (1968) "Isolation of Human Enteroviruses from Beagle Dogs," Proc. Society of Experimental Biology and Medicinet 128, pp. 463-467. Lycke, E., Magnusson, S., and Lund, E. (1965). "Studies on the Nature of the Virus Inactivating Capacity of Sea Water," Arah. ges. Virus fors chung, 17, pp. 409-413. Magnusson, S., Hedstrom, C. E., and Lycke, E. (1966) "The Virus Inactivating Capacity of Sea Water, " Acta Path. et Microbiol. Scandinav.j 66, pp. 551-559. Magnusson, S,, Gundersen, K., Brandberg, A,, and Lycke, E. (1967) "Marine Bacteria and Their Possible Relation to the Virus Inactivation Capacity of Seawater," Acta Path, et Microbiol. ScandinaVtj 71, pp. 274-280. Matossian, A, M., and Garabedian, G. A. (1967) "Virucidal Action of Sea Water," American Journal of Epidemiology, 85, pp. 1~8» McLean, D. M., and Brown, J. R. (1968) "Marine and Freshwater Virus Dispersal," Canadian Journal of Public Health, 59, pp. 100-104. Metcalf, T. G., and Stiles, W. C. (1967) "Survival of Enteric Viruses in Estuary Waters and Shellfish," in Transmission of Viruses by the Water Routet G» Berg, ed. New York: Interscience. Metcalf, T. G., and Stiles, W, C, (1968) "Viral Pollution of Shellfish in Estuary Waters," Journal Sanitary Engineering Division, ASCE, 94, pp. 595-609. Mitchell, R., and Jannasch, H. W. (1969) "Processes Controlling Virus Inactivation in Seawater," Environmental Science and Technology, 3:10, pp. 941-943. Neefe, J. R., and Stokes, J., Jr. (1945) "An Epidemic of Infectious Hepatitis Apparently Due to a Water Borne Agent," Journal American Medical As8odationt 128, pp. 1063-1075. ------- -22 Pohjanpelto, P. (1962) "Oxidation in Thermoinactivation of Polio- virus," Virologyt 16, pp. 92-94. Poynter, S. F. B, (1968) "The Problem of Viruses in Mater," Water Treatment and Examination (English), 17, pp. 187-204. Prier, J. E., and Riley, R« (1967) "Significance of Water in Natural Animal Virus Transmission" in Transmission of Viruaea by the Water Eoutet G. Berg, ed. New York: Interscience. Robeck, G. G,, Clarke, N, A., and Dostal, K, A. (1962) "Effective- ness of Water Treatment Processes in Virus Removal," Journal American Water Works Associationt 54:10, pp. 1275-1292. Romero, J. C, (1970) "The movement of Bacteria and Viruses Through Porous Media," Ground Watsrs 8:2, pp. 37-48, Shuval, H, I, (1970) "Detection and Control of Enteroviruses in the Water Environment," In Developments in Water Quality Research, H. 1, Shuval', ed. Ann Arbor: Ann Arbor-Humphreys Publishers. Shuval, H. I,tThompson, A,, Fattal, B«, Cymbalista, S., and Wiener, Y, (1971) "Natural Virus Inactivation Processes in Sea Water," Proceedings National Specialty Conference on Disinfection, Amherst, Mass., July 8-10, 1970, Stoller, A., and Collmann, R, D. (1965) "Incidence of Infective Hepatitis Followed by Down's Syndrome Sine Months'^ater»" Dec. 11, pp. 1221-1223. Taylor, F, B., Eagen, J. H., Smith, H. F. D., Jr., and Coene, R. F. (1966) "The Case for Water-Borne Infectious Hepatitis," American Journal 'Public Healthy 56:12, pp. 2093~2105, Taylor, E, W» (1967) Forty~6econd Report of the Director of Water Examination for 1965-66, Metropolitan Water Board (London). Taylor, E. W, (1969) Forty-third Report on the Results of the Bacteriological, Chemical and Biological Examination of the London Waters for 1967-68, Metropolitan Water Board (London). Trask, J. D., Vignec, A« J«, and Pa.ul, J. R, (1938) "Poliomyelitis Virus in Human Stools," Journal American Medical Association* 111, pp. 6-11. Tucker, C. B., Oven, W. H«, and Farre11, R. P. (1954) "An Outbreak of Infectious Hepatitis Apparently Transmitted Through Water," Southern Medical Journal, 47, pp. 732-740. ------- |