Heat Inactivation of Poliovirus in Wastewater Sludge

Transcription

1 APPLED AND ENVRONMENTAL MCROBOLOGY, Sept. 1976, p Copyright 1976 American Society for Microbiology Vol. 32, No. 3 Printed in U.S.A. Heat nactivation of Poliovirus in Wastewater Sludge RCHARD L. WARD,* CAROL S. ASHLEY, AND RCHARD H. MOSELEY Sandia Laboratories, Albuquerque, New Mexico 87115,* and University of New Mexico, Albuquerque, New Mexico Received for publication 26 May 1976 The effect of raw and anaerobically digested sludge on heat inactivation of poliovirus was investigated. Raw sludge was found to be very protective of poliovirus plaque-forming ability at all temperatures studied, but digested sludge had variable effects that were highly dependent upon the experimental conditions. n low concentrations and at relatively low inactivation temperatures, digested sludge is nearly as protective of poliovirus as raw sludge. However, at higher temperatures and concentrations, digested sludge caused a significant acceleration of poliovirus inactivation. The difference between the protective capability of raw and digested sludge is not due to loss of protective material, because this component is present in the solids of digested sludge as well as in those of raw sludge. nstead, the difference is due to a virucidal agent acquired during digestion. Addition of this agent to the solids of either raw or digested sludge reverses the protective potential of these solids during heat treatment of poliovirus. One of the great challenges facing our urbanized society is the disposal ofmassive quantities of wastewater sludge. Conservation of this natural resource within the food chain offers an economically feasible and highly beneficial method of sludge disposal. However, direct utilization of sludge for such purposes as cropland fertilizer or feedlot supplement is limited by the presence of human pathogens. Many of the enteric viruses found in wastewater are bound to solids and ultimately become a component of sludge (9, 10, 19). A possible method of ridding sludge of these pathogens is heat treatment. Heating sludge at high temperatures for extended periods of time is not only a costly procedure, however, but one that may destroy a large portion of its potential value. Therefore, if viruses are to be inactivated in sludge by an elevation of temperature, it is highly desirable to define an effective treatment that requires a minimal amount of heat. Viral disinfection in wastewater is commonly studied by using poliovirus as an indicator. A number of investigations have been made concerning the rate and mechanism of inactivation of this virus at various temperatures in defined media (2-4, 6-8, 20). However, the rate of heat inactivation ofpoliovirus in sludge has not been measured. Because a variety of substances protect poliovirus against heat inactivation (cf. 8, 14, 16, 17), the presence of sludge may cause a considerable reduction in its inactivation rate. The purpose of this study was to determine the 339 effects of raw and anaerobically digested sludge on the rate of heat inactivation of poliovirus. MATERLALS AND METHODS Cells and virus. Both the growth and plaquing of infectious virus were carried out on a line of HeLa cells, which was a gift of Roger Radloff (Department of Microbiology, University of New Mexico, Albuquerque). These cells were grown in monolayer cultures in Eagle medium containing 5% newborn calf serum. Three strains of poliovirus were used. The attenuated type-1 strain CHAT was obtained from the American Type Culture Collection, and the type-1 strain Mahoney and type-2 strain 712-Ch-2ab were gifts of Karl Lonberg-Holm (Central Research and Development Department, E.. dupont de Nemours & Co., Wilmington, Del.). Stock preparations of these viruses were made as described in an earlier paper (18). Heat treatment and infectivity assay of virus in sludge. Anaerobically digested sludge was obtained from the primary digester at the Albuquerque Sewage Treatment Plant, and raw sludge was collected immediately before it entered this same digester. The solids content of digested sludge ranged between 2 and 6% after being strained and blended, but raw sludge consistently contained about 2% solids. Several batches of sludge were used in these experiments. Very consistent results were obtained with different batches of raw sludge, but variations among batches probably caused the slight differences seen from one experiment to the next with digested sludge. The first step in determining the effects of sludge on heat inactivation of poliovirus was to make a 10- fold dilution of virus directly into sludge. n experi-

2 340 WARD, ASHLEY, AND MOSELEY ments requiring diluted sludge, phosphate-buffered saline (PBS) was the diluent. After virus was mixed with sludge for 15 min at room temperature, the sample vials were flamed to eliminate infectious virus that might have remained above the water line in the incubation bath during treatment. Samples were then incubated for the times and at the temperatures specified and immediately placed in an ice bath. Before infectivity was measured, each sample was sonicated at room temperature for 2 min in 0.1% sodium dodecyl sulfate (SDS). The number of plaque-forming units in each sample was then determined as previously described (18). ndigenous viruses were beyond the limits of detection using these techniques. Therefore, the plaque-forming units found in these experiments could only have been due to seeded virus. Analysis of radioactively labeled poliovirus after treatment in sludge. Poliovirus strain CHAT was labeled either with [3H]uridine or ['4C]protein hydrolysate and extensively purified as described in a previous publication (18). Samples of these preparations were mixed with sludge, heat treated, and prepared for analysis by the SDS-sonication procedure as described above. Before the effects of this treatment on radioactively labeled virus particles were examined, sludge solids were removed by centrifugation at 18,000 x g for 20 min. Recovery of labeled viruses in the supernatant fraction was determined by measuring a portion of each sample for both total radioactivity and radioactivity precipitable with 5% trichloroacetic acid. Total radioactivity was measured in Bray scintillation fluid, whereas acid-precipitable radioactivity was collected on membrane filters and measured in toluene-based scintillation fluid. Sedimentation analysis of labeled viruses was then carried out by centrifugation in glycerol gradients as previously described (18). RESULTS nfectious polioviruses are fully recoverable from sludge. The rate of heat inactivation of poliovirus in sludge cannot be critically studied unless full recovery of infectious virus can be obtained. The technique used to recover virus from seeded sludge was to sonicate in the presence of 0.1% SDS and directly analyze for plaque-forming units. The use of SDS and sonication was found to cause no detectable change in the plaque-forming ability ofpoliovirus. Furthermore, simnply mixing virus with raw sludge has no effect on the recovery of plaque-forming units for the three strains of poliovirus studied with this technique (Table 1). However, a loss of recoverable infectivity of between 25 and 50% invariably occurred in each of more than 10 replicate experiments after one of the three strains of virus (strain CHAT) was mixed with digested sludge. A detailed study (18) established that this loss is not caused by the inability to detect infectious virus but is due to viral inactivation by a component of digested sludge. Therefore, viruses that remain infectious after mixing with sludge are fully recoverable by the techniques used here. Heat-induced loss of poliovirus plaqueforming units in sludge. The effect of raw and anaerobically digested sludge on the rate of loss of poliovirus (strain CHAT) plaque-forming units as a function of temperature was investigated. Raw sludge is quite protective of the virus at all temperatures studied here (Fig. 1). On the other hand, the rate of loss of plaqueforming units in digested sludge relative to that occurring in buffer alone is dependent upon the temperature. At the lowest temperature studied (43 C), digested sludge is somewhat protective, but at the highest temperature used (51 C) digested sludge accelerates loss of titer. Probably the most significant feature of the data presented in Fig. 1 is the dramatically different effects of raw and digested sludge in these experiments. The apparent explanations for this difference are that raw sludge either contains a protective substance that is lost upon digestion or acquires an activity during digestion that accelerates the rate of heat inactivation of poliovirus. However, a third explanation cannot be overlooked. Digested sludge may contain a component that is stimulated by heat to become strongly bound to poliovirus and, in so doing, mask the plaque-forming ability of otherwise infectious viruses. This latter possibility can be ruled out if it can be shown that the loss of poliovirus titer with heat in digested sludge is due to viral inactivation. Therefore, the physical nature of virus particles after heat treatment in digested sludge was investigated. Breakdown of poliovirus during heat treatment in anaerobically digested sludge. The nature of poliovirus particles after heat treat- TABLE 1. Recovery of three strains ofpoliovirus from seeded sludge Recovery of plaque-forming units Sample Strain CHAT Strain Mahoney Strain 712 No sludge (PBS) 2.3 x x x 108 Raw sludge 2.3 x 10, (100)a 9.1 x 108 (100) 2.8 x 108 (104) Digested sludge 1.7 x 108 (74) 9.5 x 108 (104) 2.7 x 108 (100) a Numbers in parentheses are percentage of recoveries relative to control. APPL. ENVRON. MCROBOL.

3 VOL. 32, 1976 ment in digested sludge was studied by the use of purified, radioactively labeled virus. For this, digested sludge was seeded with labeled virus, heated at 43 C for 200 min, and prepared for analysis by sonication in 0.1% SDS. This treatment was found to cause about a 3-log decrease in recoverable plaque-forming units (see Fig. 1). After heating, sludge solids were removed from the samples by centrifugation at 18,000 x g for 20 min, and virus retained in the supernatant fraction was analyzed. The initial experiment was designed to determine virus recovery. By measurement of total radioactivity, it was found that some radioactive material (15%) is removed with the solids when the virus is labeled with [14C]protein hydrolysate (Table 2). However, when the particles are labeled with [3H]uridine, radioactivity LL10 ~ ~ ~ ~ ~ - io'-23 L. 1-U 10 z HEAT NACTVATON OF POLOVRUS 341 is recovered in full. The difference between the percentage of recovery of viral RNA and viral protein can be explained by examination of the recovery of acid-precipitable radioactivity. Very little of the labeled viral RNA remains acid precipitable after heat treatment of poliovirus particles in digested sludge, whereas almost one-half of the recoverable viral protein is still large enough to be precipitable with acid (Table 2). This result indicates that poliovirus particles are broken down during heat treatment and that their ribonucleic acid molecules are hydrolyzed and released into the medium. However, viral proteins are less extensively degraded than viral ribonucleic acid, and a small percentage of these protein molecules apparently remains associated with sludge solids during centrifugation. TME (MNUTES) FG. 1. Effect of sludge on the rate of heat inactivation of poliovirus. After a 10-fold dilution into buffer (PBS), raw sludge, or anaerobically digested sludge, poliovirus strain CHAT was mixed for 15 min at room temperature and heated at the specified temperature. Samples were removed from the incubation bath at the times shown and immediately cooled in ice. After sonication in 0.1% SDS, each sample was directly assayed for total plaque-forming units on HeLa cells. Symbols: 0, raw sludge; *, digested sludge; 0, PBS. TABLE 2. Recovery ofradioactively labeled poliovirus strain CHAT in digested sludge supernatant after heat treatment Radioactivity recovered (cpm) Radioactive Label Sample Total Acid precipitable [14C]protein hydrolysate Unheated control (PBS) 1,685 1,602 Heated for 200 min, 43 C, in digested 1,432 (85.0)a 703 (43.9) sludge [3H]uridine Unheated control (PBS) 7,145 5,182 Heated for 200 min, 43 C, in digested 7,287 (102) 606 (11.7) sludge a Numbers in parentheses are percentage of recoveries relative to unheated control.

4 342 WARD, ASHLEY, AND MOSELEY The conclusion that breakdown of poliovirus particles occurs in digested sludge during heat treatment was confirmed by a second experiment. Here, the sedimentation coefficient of radioactively labeled virus was measured. Labeled particles have much lower sedimentation values after than before heat treatment (Fig. 2). Thus, poliovirus is broken down and irreversibly inactivated when heated in digested sludge. Effects of different concentrations of sludge on heat inactivation of poliovirus. n the experiments presented above, undiluted sludge was seeded with poliovirus before heat treatment. n an attempt to determine the dif- m 0- -a, (-) r APPL. ENVRON. MCROBOL. ference between the effects of raw and anaerobically digested sludge seen in these experiments, the rate of heat inactivation of poliovirus was studied after seeding lower concentrations of sludge. The first experiment was designed to quantitate the inactivation of strain CHAT during 200 min at 43 C over a wide range of concentrations ofdigested sludge. The results were quite unexpected. Extremely small amounts of sludge are highly protective of the virus (Fig. 3). However, this protection diminishes as the concentration of sludge is increased. At the highest concentration used, digested sludge is almost as unprotective as PBS. BEFORE A~~~~~- lafter.~~~~~~~~~~~~~ ~~~~~~~~~~~~~ 0 1 a~~~~~~~~ & ~~~~~~~~~~~~~~~~~ 0 t p n-' FRACTON NUMBER FG. 2. Sedimentation profiles of radioactively labeled poliovirus before and after heat treatment in anaerobically digested sludge. Purified virus (strain CHAT) labeled with [14C]protein hydrolysate was diluted 10-fold with digested sludge and mixed for 15 min at room temperature. One-half of the sample was further incubated at 43 C for200 min. After sonication in 0.1% SDS, both samples were centrifuged at 18,000 x g for20 min, and the supernatant fractions ofeach were analyzed by sedimentation in density gradients (15 to 30% glycerol, SW27.1 rotor, 27,000 rpm, 2.5 h, 4 C). Fractions were collected from the bottom of the gradients and measured for total radioactivity. 1!

5 VOL. 32, 1976 HEAT NACTVATON OF POLOVRUS 343 To e,xamine the effect of sludge concentration concentrations, but this capability is especially in greiater detail, heat studies were carried out evident in the greatest concentration at the with ail three strains of poliovirus in low and highest temperatures (Table 3). Digested high concentrations of both raw and digested sludge, on the other hand, is significantly prosludge Raw sludge is quite protective at both tective only at relatively low temperatures and concentrations. These results indicate that the protective component of raw sludge is quite active even CL when present in very low concentrations. Because the same low concentrations of 01 digested and raw sludge are almost equally protective at 10 the lowest temperatures studied, this component of raw sludge appears to be retained after 0: z digestion. At higher sludge concentrations and C-) temperatures, the expression of the protective component seems to be limited by another substance found only in digested sludge. Thus, the 0n difference between raw and digested sludge in these experiments may be due to a virucidal activity acquired during digestion. The agent responsible for this activity is possibly the same sludge component previously shown to cause poliovirus inactivation at much lower (ML/2ML SAMPLE VOLUME) temperatures than those used here (18). FG. 3. Survival ofpoliovirus after heat treatment Physical separation of sludge components in var ous concentrations of anaerobically digested affecting heat inactivation of poliovirus. sludge. Be-. Poliovirus strain CHAT was diluted 10-fold cause anaerobically digested sludge exhibits into PB;S that contained the specified amounts of two activities having opposite on the digeste!d sludge. After mixing for 15 min at room o act ivavmg ofpolioveffectshould temperature, samples were heated at 43 C for 200 rate of heat inactivation of poliovirus, it should min, s4onicated in 0.1% SDS, and assayed for total be possible to physically separate the compoplaque--forming units. nents responsible for these activities. This was TABLz 3. Effect of sludge concentration on heat inactivation ofpoliovirus % Survival of PFUb Samplea Treatment Strain Ma- Strain CHAT Strhoney Strain 712 No sludge 39 C, 200 min ml of raw sludge ml of raw sludge ml of digested sludge ml of digested sludge No sludge 4300, 200 min ml of raw sludge ml of raw sludge ml of digested sludge ml of digested sludge No sludge 4700, 20 min ml of raw sludge ml of raw sludge ml of digested sludge ml of digested sludge No sludge 5100, 5 min ml of raw sludge ml of raw sludge ml of digested sludge ml of digested sludge < a Each sample contained 0.2 ml of poliovirus lysate, the specified volume of sludge, and the remainder as PBS in a total volume of 2.0 ml. b Survival was determined relative to unheated control in PBS. PFU, Plaque-forming units.

6 344 WARD, ASHLEY, AND MOSELEY attempted by fractionation of sludge into solids and liquid through centrifugation (18,000 x g, 20 min) and comparison of the virucidal and protective capacity of each to that of unfractionated sludge. When the solids from a small amount of either digested or raw sludge are resuspended in PBS, their protective capabilities are very similar to that of unfractionated sludge containing an identical concentration of solids (Table 4). n contrast, an equivalent volume of the liquid fraction from raw or digested sludge was found to be totally unprotective in this experiment. These results show that the solids of both raw and digested sludge contain most of the protective component. They also support the previous conclusion that raw sludge retains its protective capability after anaerobic digestion. The fraction of digested sludge that contains the virucidal component was then determined. Because this agent is much more readily expressed when present in high concentrations, heat inactivation of poliovirus was studied in fractionated samples from undiluted sludge. Most of the virucidal activity is removed with the liquid portion of digested sludge (Table 4). However, even when the solids of digested sludge are washed several times with PBS to remove this component, these solids are still not as protective as those of raw sludge. n addition, a high concentration of digested sludge solids is not as protective as a low concentration. Therefore, it appears that a small TABLE 4. Survival ofpoliovirus strain CHAT after heat treatment in fractionated sludge % Survival of PFUa after 200 Sludge Sample min at 43 C concn Digested Raw sludge sludge No sludge PBS 0.03 Lowb Total sludge Sludge supernatant Resuspended solids (PBS) Highe Total sludge Sludge supernatant Resuspended solids (PBS) (first centrifugation) Resuspended solids (PBS) 5.5 NDd (second centrifugation) Resuspended solids (PBS) 6.3 ND (third centrifugation) a PFU, Plaque-forming units. beach sample contained either 0.05 ml of total sludge, the solids from this volume of sludge, or an equal volume of digested sludge liquid in a total sample volume of 2 ml. c Samples contained 1.8 ml of either total or fractionated sludge per 2-ml sample volume. d ND, Not determined. APPL. ENVRON. MCROBOL. amount of virucidal activity is retained with this fraction of digested sludge. t should be noted that heat inactivation of poliovirus under the conditions studied here is equally effective in PBS and in undiluted supernatant from digested sludge. Because this sludge fraction contains most of the virucidal agent, a greater amount of inactivation was expected in it than in PBS. The apparent explanation for this result is found upon examination of the protective capability of a high concentration of raw sludge supernatant. This quantity of raw sludge supernatant is highly protective (Table 4). Therefore, some protective material is retained in the liquid fraction of raw sludge and is probably also still present in the supernatant of digested sludge. From this it appears that the actual rate of heat inactivation of poliovirus in digested sludge supernatant, as in other fractions of digested sludge, is determined by a competition between protective and virucidal components. Taken together, these results conclusively demonstrate that anaerobically digested sludge contains both protective and virucidal components and that these components can, for the most part, be physically separated by the fractionation of sludge solids and liquid through centrifugation. Reversal of raw sludge protection with the virucidal agent of digested sludge. The solids of both raw and digested sludge are highly protective of poliovirus during heat treatment, but inactivation occurs much more rapidly in digested than in raw sludge because a virucidal agent is acquired during digestion. Because this agent is found primarily in the liquid portion of digested sludge, it may be possible to resuspend the solids of raw sludge in this liquid and reverse the stabilizing effect on poliovirus normally provided by these solids. This is indeed the case for all three strains of poliovirus tested. The amount of heat inactivation of poliovirus that occurs during 5 min at 51 C in raw sludge is much greater in the presence than in the absence of the virucidal agent (Table 5). n fact, the amount of inactivation approaches that observed in digested sludge under these conditions. Therefore, once this agent has been identified, its addition to raw sludge should significantly reduce the heat requirements needed to inactivate poliovirus and possibly accelerate the inactivation of other viruses. DSCUSSON Although viruses are readily inactivated by heat, the rate of inactivation is highly depend-

7 VOL. 32, 1976 TABLE 5. Effect of virucidal activity on heat inactivation ofpoliovirus in raw sludge Sample % Survival of PFUa after 5 min at 51'C Strain Mahoney Strain 712 PBS Digested sludge Raw sludge Raw sludge solids resus pended in digested sludge supernatant a PFU, Plaque-forming units. ent on environment. Sludge provides an environment that could have drastic effects on viral inactivation rates. This study was designed to determine the effects of raw and anaerobically digested sludge during heat inactivation of three different strains of seeded poliovirus. Raw sludge was found to be consistently protective of poliovirus during heat treatment, but digested sludge appears to have very erratic effects. When at low inactivation temperatures and very dilute, it is protective of poliovirus. However, at high temperatures and sludge concentrations, loss of viral plaque-forming units is more rapid in digested sludge than in buffer. Differences between raw and digested sludge that would account for these results were then sought. One possible difference was that digested sludge may contain a component that, at high temperatures, binds strongly to virus particles and masks their infectivities. This possibility was eliminated upon examination of radioactively labeled poliovirus after heat treatment in digested sludge. Such treatment causes the sedimentation values of labeled particles to be considerably reduced while, at the same time, the RNA molecules of these degraded particles become almost totally acid soluble. Thus, loss of infectivity is not due to masking but to irreversible inactivation of poliovirus. This finding led to the conclusion that the difference between raw and digested sludge is caused either by the loss of a protective component or by the acquisition of virucidal activity during digestion. Because digested sludge in low concentrations and at low inactivation temperatures is nearly as protective of poliovirus as raw sludge, loss of a protective component did not appear to be the explanation. This conclusion is supported by the finding that a high concentration of digested sludge solids is very protective of virus when the liquid fraction of sludge has been removed. From these results it appears that a virucidal HEAT NACTVATON OF POLOVRUS 345 agent that is mainly associated with the liquid portion of sludge is produced during digestion. As expected, the liquid fraction of digested sludge retains the virucidal activity, whereas no such activity is detectable in the liquid fraction of raw sludge. Therefore, digested sludge has not lost the protective component, but, instead, the expression of this component is muted under certain conditions by a virucidal agent acquired during digestion. Another environmental system that has been found to contain two components with opposite effects on virus survival is seawater. Although seawater has been known to possess a virucidal capability for some time (1, 11-13, 15), a limited separation of protective and virucidal components from seawater has only recently been carried out (5). Possible similarities between the virucidal component of digested sludge and seawater have not been investigated. We have previously shown that anaerobically digested sludge, but not raw sludge, contains a virucidal agent that inactivates poliovirus at much lower temperatures than those studied here (18). t is quite likely that this agent is also responsible for accelerating heat inactivation of poliovirus. Confirmation of this suggestion awaits further characterization of this sludge component. Although the solids of raw sludge are extremely protective during heat inactivation of poliovirus, this protection can be reversed by the addition of the liquid fraction of digested sludge. As a result, the temperature and time required for inactivation can be considerably reduced. Thus, it should be possible to heat inactivate poliovirus in raw as well as in digested sludge under rather mild conditions. The ability of the virucidal agent to reduce the heat required to inactivate other viruses is being investigated. ACKNOWLEDGMENTS We thank C. A. Trauth, Jr., and K. S. Neuhauser of Sandia Laboratories for helpful discussions and constructive criticism of the manuscript. This investigation was supported by the Division of Nuclear Research and Applications, U.S. Energy Research and Development Administration, and the Environmental Protection Agency under nteragency Agreement E (29-2)-3536/ EPA-LAG LTERATURE CTED 1. Akin, E. W., W. F. Hill, G. B. Cline, and W. H. Benton The loss of poliovirus 1 infectivity in marine waters. Water Res. 10: Breindl, M The structure of heated poliovirus particles. J. Gen. Virol. 11: Dimmock, N. J Differences between the thermal inactivation of picornaviruses at "high" and "low" temperatures. Virology 31: Dugan, V. L., and R. Trujillo Heat-accelerated

8 346 WARD, ASHLEY, AND MOSELEY radioinactivation of attenuated poliovirus. Radiat. Environ. Biophys. 12: Gerba, C. P., and G. E. Schaiberger Effects of particulates on virus survival in seawater. J. Water Pollut. Control Fed. 47: Hinuma, Y., S. Katagiri, M. Fukuda, K. Fukushi, and Y. Watanabe Kinetic studies on the thermal degradation of purified poliovirus. Biken J. 8: Koch, G nfluence of assay conditions on infectivity of heated poliovirus. Virology 12: Lonberg-Holm, K., L. B. Gosser, and J. C. Kauer Early alteration of poliovirus in infected cells and its specific inhibition. J. Gen. Virol. 27: Lund, E Observation on the virus binding capacity of sludge, p. 1-24, 1-5. n S. H. Jenkins (ed.), Advances in water pollution research, Proceedings of the Fifth nternational Conference, San Francisco. Permagon Press, New York. 10. Lund, E., and V. Ronne On the isolation of virus from sewage treatment plant sludges. Water Res. 7: Lycke, E., S. Magnusson, and E. Lund Studies on the nature of the virus inactivating capacity of seawater. Arch. Gesamte Virusforsch. 17: Magnusson, S., C. E. Hedstrom, and E. Lycke APPL. ENVRON. MCROBOL. The virus inactivating capacity of seawater. Acta pathol. Microbiol. Scand. 66: Matossian, A. M., and G. A. Garabedian Virucidal action of seawater. Am. J. Epidemiol. 85: Pohjanpelto, P Stabilization of poliovirus by cystine. Virology 6: Shuval, H.., A. Thompson, B. Fattal, S. Cymbalista, and Y. Wiener Natural virus inactivation processes in seawater. J. Sanit. Eng. Div. Am. Soc. Civ. Eng. SA5: Steele, F. M., and F. L. Black nactivation and heat stabilization of poliovirus by 2-thiouracil. J. Virol. 1: Wallis, C., and J. L. Melnick Cationic stabilization-a new property of enteroviruses. Virology 16: Ward, R. L., and C. S. Ashley nactivation of poliovirus in digested sludge. Appl. Environ. Microbiol. 31: Wellings, F. M., A. L. Lewis, and C. W. Mountain Demonstration of solids-associated virus in wastewater and sludge. Appl. Environ. Microbiol. 31: Youngner, J. S Thermal inactivation studies with different strains of poliovirus. J. mmunol. 78: Downloaded from on December 7, 2018 by guest