Stability of Rotavirus

Transcription

1 APPLED AND ENVRONMENTAL MCROBOLOGY, June 1980, p Vol. 39, No /80/ /05$02.00/0 Effects of Wastewater Sludge and ts Detergents on the Stability of Rotavirus RCHARD L. WARD'* AND CAROL S. ASHLEY2 Division 4535, Sandia Laboratories, Albuquerque, New Mexico and University of New Mexico, Albuquerque, New Mexico Wastewater sludge reduced the heat required to inactivate rotavirus SA-11, and ionic detergents were identified as the sludge components responsible for this effect. A similar result was found previously with reovirus (R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol. 36: , 1978). The quantitative effects of individual ionic detergents on rotavirus and reovirus were very different, and rotavirus was found to be extremely sensitive to several of these detergents. However, neither virus was destabilized by nonionic detergents. On the contrary, rotavirus was stabilized by a nonionic detergent against the potent destabilizing effects of the ionic detergent sodium dodecyl sulfate. The destabilizing effects of both cationic and anionic detergents on rotavirus were greatly altered by changes in the ph of the medium. Rotaviruses are enteric viruses which are released in large numbers in the feces of infected animals and humans. Because these viruses are believed to cause at least 50% of the cases of enteritis among infants throughout the world (1), they are some of the most important pathogens in wastewater and wastewater sludges. To prevent the spread of these viruses, it is important to find the most feasible methods to inactivate rotaviruses in sludge. Because rotaviruses have physical and chemical characteristics similar to those of reovirus (7) but have significant antigenic and morphological differences (2-6), it has been proposed that they be classified as a separate genus within the family Reoviridae. Reovirus is the prototype of the viruses in this family and has previously been chosen as the representative for inactivation studies in wastewater sludges. These studies have shown that the ionic detergents in sludge destabilize reovirus and reduce the heat required for its inactivation (9, 10). They have also shown that the degree of destabilization is highly dependent on ph. This is one apparent cause for the more rapid inactivation of reovirus observed in sludge at low and high ph values as compared with ph values near neutrality (11). Because of technical difficulties, it is not yet feasible to study the inactivation of human rotaviruses in sludge. However, it is now possible to carry out this type of study with animal rotaviruses. Therefore, a series of inactivation experiments in wastewater sludge were performed with SA-1l, a rotavirus strain isolated from a vervet monkey. These experiments can be directly compared with those previously car ried out with reovirus. As shown below, ionic detergents in sludge destabilize SA-1 in a manner similar to that observed with reovirus. However, the efficiency with which different detergents cause destabilization varies for the two viruses. MATERALS AND METHODS Cells and virus. Simian rotavirus strain SA-11 was used throughout this study. The virus was grown and assayed for infectivity in monolayer cultures of CV-1 cells grown in Eagle minimal essential medium (MEM) supplemented with 10% fetal calf serum. The virus and cells were both gifts from C. P. Gerba, Baylor College of Medicine, Houston, Tex. Although originally contaminated with mycoplasmas, the cells were freed of these contaminants by L. C. McLaren (University of New Mexico School of Medicine, Albuquerque) through extensive treatment with gentamicin. Production of rotavirus in CV-1 cells. Cells were washed three times with MEM without serum and infected with rotavirus at a multiplicity of infection of 10 to 20 plaque-forming units per cell. After an adsorption period of 30 min at 37 C, the infected monolayers were overlaid with MEM without serum and incubated for an additional 24 to 48 h (until cytopathic effects were complete). Cells and medium were combined, and samples were stored frozen at -60 C. The infectivity of this material was typically 2 x 107 plaqueforming units per ml. Plaque assay for rotavirus SA-11. The infectivity of rotavirus SA-11 was determined by the plaque assay. The procedure used was similar to that described by Smith et al. (8), with several modifications. Confluent monolayers of CV-1 cells in 60-mm plastic petri dishes were washed three times with MEM without serum. Viruses in this same medium were added (0.2 ml) and allowed to adsorb for 30 min at 37'C with occasional rocking of the plates. Soft agar overlay

2 Voit,.,39, 1980 mediu1m (4 ml), also at 37 C, was then added. This medium (MEM) contained 0.3% Noble agar, 10% tryptose phosphate broth, 25 Mg of diethylaminoethyl dextran per ml, and 2 [Lg of trypsin per ml. No serum was included in this medium. After 4 days at 37 C, the soft agar was removed, and plaques were counted after direct staining of the cell monolayers with 0.5% crystal violet. Treatment of virus with sludge, sludge components, and detergent solutions. Rotavirus (5 x io) plaque-forming units per ml) was diluted 10-fold into anaerobicallv digested sludge (5% [wt/wt] solids), into fractionated samples of this sludge, or into solutions of specific detergents to determine the effects of these materials on this virus. Samples were incubated in a water bath for different times and temperatures. After treatment, these samples were sonicated at room temperatuire for 2 min just before serial dilution in METM and analvsis for recoverable infectivity. RESULTS Effects of sludge and sludge fractions on rotavirus. The effect of wastewater sludge on the stability of rotavirus SA-li was first determined through a comparison of the amounts of viral inactivation during 15 min of heat treatment in water and sludge. No significant inactivation of SA- 11 occurred in water until the temperature was maintained at about 50 C, but viral infectivity decreased by more than three orders of magnitude during 20 min at 55 C (Fig. 1). n sludge, comparable effects were observed, but at temperatures that were about 5 C lower. A simliar result was reported previously with reovirus, except that sludge caused a greater reduction in the heat required to inactivate this enteric virus (9) Ṫhe agents in sludge responsible for the decreased heat stability of reovirus were isolated -t Temperatuwr (OC) FG. 1. Heat inactitation ofrotavirus in uwater and in sludge. Rotauirus preparations were diluted 10- fold into either water (-) or anaerobically digested sludge (-), heated for 15 min at different temperatures, and assayed for plaque-forming units (pfu). STABLTY OF ROTAVRUS 1155 by a series of purification procedures and identified as ionic detergents (10). When the same sludge samples isolated during these procedures were tested for their effects on rotavirus, the results were quite similar to those found with reovirus (Table 1); that is, the original sludge had some activity under the conditions tested, but this activity increased greatly upon purification. t was concluded, therefore, that sludge contains components that stabilize both reovirus and rotavirus, but that these are removed during purification of the components with destabilizing activity. Because the components isolated from sludge that are responsible for destabilization of reovirus have been identified as ionic detergents (10) and because detergents were copurified with the sludge components that destabilized rotavirus (Table 1), it is possible that these components are also detergents. Effects of detergents on the stability of rotavirus. The following experiments were designed to determine whether detergents do in fact cause rotavirus inactivation and, if so, which types of detergents have this effect. The effect of sodium dodecyl sulfate (SDS), a standard anionic detergent, was examined first. SDS effectively inactivated SA-li during a period of 60 min at ph 7 (Fig. 2), even at room temperature (21 C) and at quite low concentrations (0.01 to 0.1%). The conditions under which SDS was shown to be virucidal for rotavirus were then used to examine the effects of other detergents and detergent-like compounds on this virus (Table 2). As was found with reovirus (10), the hydrophobic regions of these compounds must have a crucial minimal size to cause rotavirus inactivation. For example, SDS was active whereas sodium decyl sulfate was not, and dodecyltrimethylammonium chloride was active, but nonyltrimethylammonium bromide was not. Also, as found with reovirus, nonionized detergents showed no virucidal activity, whereas both positively charged and negatively charged compounds inactivated rotavirus. n contrast to the results previously found with reovirus (10), several of the ionic detergents did not inactivate rotavirus (lauroyl sarcosine, Sarkosyl 0, Standapol ES-40, Alipal Co-436) (Table 2). n fact, these detergents and the nonionic detergents tested (Ethosperse LA-4 and gepal Co-630) reproducibly caused increases in rotavirus infectivity under these conditions. The most probable causes for these increases in viral infectivity by detergents are dissociation of viral aggregates and removal of some virion-associated material that blocks infectivity. To determine whether these compounds were

3 1156 WARD AND ASHLEY APPL. ENVRON. MCROBOL. TABLE 1. Heat inactivation of rotavirus and reovirus and recovery of anionic detergents in samples of fractionated sludge % Recovery after treatmentb % Recovery Sample Purification step' of anionic Rotavirus' Reovirus" detergents' Control [tris(hydroxymethyl) aminomethane buffer] 1 Original sludge Pellet fraction from centrifugation Supernatant fraction from washed pellet of sample 2 4 Dry, heat, resuspend and centrifuge sample Centrifuge sample 4 at ph 2 and See reference 10 for details of purification. bdetermined from assays of plaque-forming units. c Heat treatment was at 40 C for 20 min at ph 8.5 in 0.1 M tris(hydroxymethyl)aminomethane buffer. d Heat treatment was at 45 C for 10 min at ph 8.5 in 0.1 M tris(hydroxymethyl)aminomethane buffer. 'The detergent concentration was determined by the methylene blue technique. See reference 10 for details. CL 01, c e 102. \ Concentration of SDS (%) FG. 2. Effect ofsds on the infectivity ofr-otavirus. Rotavirus was diluted 10-fold into SDS at different concentrations containing 0.1 M tris(hydroxymethyl)aminomethane (ph 7), incubated for 60 min at 21 C (O) or 37 C (0), and assayed for recoverable plaque-forming units (pfu). truly ineffective against rotavirus, several were examined under more severe conditions (400C, 20 min). The ionic detergents tested under these new conditions (lauroyl sarcosine and Standapol ES-40) did accelerate SA-li inactivation (Table 3). However, the two nonionic detergents tested continued to cause an increase in infectivity at this higher temperature. These results suggest that most, if not all, ionic detergents have qualitatively similar effects on rotavirus and reovirus. However, the quantitative effects of these detergents are clearly quite different for these two viruses. The increases in infectivity of rotavirus observed after treatment with nonionic detergents at both 21 and 40 C suggested that these detergents might in fact stabilize this virus. To test this possibility, we measured the effect of a constant concentration of gepal Co-630 (0.1%) on the heat inactivation of rotavirus at several concentrations of SDS. At a concentration of 0.01%, SDS caused efficient inactivation of rotavirus, but the presence of 0.1% gepal effectively protected the virus (Table 4). However, 0.1% gepal could not prevent rotavirus inactivation by 0.1% SDS. Therefore, a nonionic detergent can stabilize rotavirus and partially, but not totally, overcome the virucidal effects of an ionic detergent. ph modification of the effects of detergents on rotavirus. The studies reported above concerning the effects of individual detergents on rotavirus were all carried out at ph 7. n a previous report (11), it was demonstrated that changes in ph dramatically alter the effects of detergents on other viruses. Therefore, the effects of two representative ionic detergents on rotavirus were studied as a function of ph. The detergents chosen for this study were SDS, an anionic detergent, and dodecyltrimethylammonium chloride, a cationic detergent. Although SDS was quite virucidal at all ph values examined, a minimal effect on rotavirus was found near neutrality, and more inactivation was observed in an acidic medium than in an alkaline medium (Fig. 3). Dodecyltrimethylammonium chloride increased the amount of rotavirus inactivation at all ph values examined, but

4 VOL. 39, 1980 STABLTY OF ROTAVRUS 1157 TABLE 2. Effects of detergents and detergent-like compounds on the infectivity of rotavirus % Recovery of vi- Compound Structural formula rus after 60 min at 210C1 Control [tris(hydroxymethyl)- 100 aminomethane buffer] Sodium octyl sulfate CH3(CH2)7OSO3:Na+ 75 (100) Sodium decyl sulfate CH3(CH2)9OSO3FNa+ 85 (59) SDS CH3(CH2)OSO3Na+ <0.025 (0.003) Sodium dodecyl benzene sul- CH3(CH2),C6H4SO3-Na+ <0.025 (<0.001) fonate p-toluenesulfonic acid CH3C6H4SO3-100 (100) Lauroyl sarcosine CH3(CH2),oCON(CH:,)CH2COO- 290 (0.003) Sarkosyl Ob CH3(CH2)5CH=CH(CH2)rCON(CH3)CH2COO- 260 (0.002) Standapol ES-40C CH3(CH2)130CH2CH20SO:3-270 (0.4) Alipal Co-436d CH3(CH2)8C6H40(CH2CH20)4SO3-230 (0.01) Dodecyltrimethylammonium CH3(CH2)11N+(CH3)3Cl- 5.0 (<0.001) chloride Nonyltrimethylammonium CH3(CH2)8N+(CH3):,Br- 90 (97) bromide BTC-824 P-100e CH3(CH2)13N+(CH:,)2CH2C6H. Cl- <0.025 (<0.001) Ethosperse LA-4f CH3(CH2)110(CH2CH20)5H 170 (100) gepal Co-630d CH3(CH2)8C6H40(CH2CH20)sH 210 (100) a Rotavirus was diluted 10-fold into a 0.1% solution of each compound in 0.1 M tris(hydroxymethyl)- aminomethane (ph 7.0), left for 60 min at 21 C, and assayed for recoverable plaque-forming units. Numbers in parentheses are values for reovirus recoveries after 20 min at 45 C (see reference 10 for details). b Gift from Geigy Chemical Corp. 'Gift from Standard Chemical Products. d Gift from GAF Corp. 'Gift from Onyx Chemical Co. f Gift from Glyco Chemical nc. TABLE 3. Effect of heat on rotavirus inactivation in the presence of selected ionic and nonionic detergents % Recovery of virus after 20 Compound min at 40'C' Control [tris(hydroxy- 100 methyl)aminomethane buffer] Lauroyl sarcosine 10 Standapol ES Ethosperse LA gepal Co a Rotavirus was diluted 10-fold into 0.1% solutions of each compound in 0.1 M tris(hydroxymethyl)- aminomethane (ph 7.0) at 4 C and incubated at 40 C for 20 min before being assayed for recoverable plaqueforming units. its effect was much greater at alkaline ph's. This result is similar to that previously observed with reovirus (11). However, it should be noted that inactivation of rotavirus by both SDS and dodecyltrimnethylammonium chloride occurred at much lower temperatures at most ph values than was found with reovirus. DSCUSSON We have carried out a number of studies concerning the effects of wastewater sludges on TABLE 4. Stabilization of rotavirus against SDS by a nonionic detergent Treatment medium' % Recovery of virus after 20 min at 400CG 0.1 M Tris (ph 7) M Tris (ph 7) % SDS M Tris (ph 7) % SDS M Tris (ph 7) + 0.1% SDS M Tris (ph 7) + 0.1% gepal 130 Co M Tris (ph 7) + 0.1% gepal % SDS 0.1 M Tris (ph 7) + 0.1% gepal % SDS 0.1 M Tris (ph 7) + 0.1% gepal % SDS a Tris, tris(hydroxymethyl)aminomethane. brotavirus was diluted 10-fold at 4 C with the combinations of buffer and detergents indicated, incubated for 20 min at 40 C, and assayed for recoverable plaque-forming units. enteric viruses. n this report the effects of sludge on rotavirus, an important enteric pathogen, were investigated. t was shown that sludge can reduce the amount of heat required to inactivate rotavirus and that at least one group of agents responsible for this effect is the ionic detergents. Furthermore, we showed that

5 1158 WARD AND ASHLEY lill w= 103. l A D \, #'i\ #, U,' - 'A A ph FG. 3. Effect of ph on rotavirus inactivation by detergents. Rotavirus was diluted 10-fold into 0.1 M solutions of acetate (ph 2 and 4), phosphate (ph 6), tris(hydroxymethyl)aminomethane (ph 8), or borate (ph 10) buffer which contained no detergent (0), 0.1% SDS (U), or 0.1% dodecyltrimethylammonium chloride (A). After incubation at either 21 C (A) or 4 C (B) for 60 min, the final ph of each sample was measured, and the number of infectious viruses was determined by the plaque assay. pfu, Plaque-forming units. the effects of model cationic and anionic detergents on this virus were modified by changes in ph. Similar observations were reported previously for reovirus, the prototype for viruses belonging to the Reoviridae (9-11). This finding suggested that reovirus may be an appropriate model for environmental studies with viruses belonging to this family. The finding that reovirus and rotavirus show the same qualitative responses but dissimilar quantitative responses to individual ionic detergents demonstrates that molecular variations among members of this virus family do cause some differences in their responses to environmental factors. However, similar differences could probably be found between different strains within the same virus group. n any event, these differences are not nearly as great as those already reported between reovirus and the enteroviruses (11). n that study it was shown that SDS and dodecyltrimethylammonium chloride stabilize enteroviruses against heat inactivation at ph values between 6 and 9, a very different result from that found with either reovirus or rotavirus. Although there may be a number of compounds in sludge that can stabilize rotavirus, nonionic detergents, which are known ingredients of sewage, were shown here to have this stabilizing effect. t is possible that removal of nonionic detergents from sludge caused the destabilizing effects of ionic detergents to be better expressed after their separation from other sludge components. Why nonionic and ionic detergents have such different effects on rotavirus APPL. F' NVRON. MCROBOL. is not evident from chemical considerations, nor is it clear why slight structural alterations in an ionic detergent can cause it to have very different effects on this and other viruses. Certainly the differences between the effects of charged and uncharged detergents may reflect differences in the extents to which the two detergent types interact with the virus particles and the nature of the interaction. One method that can be used to help understand the cause of these different effects is to examine the molecular changes produced in virus particles by chemical agents. Such a study is the subject of the accompanying paper, in which we describe the effects of SDS and the chelating agent ethylenediaminetetraacetate with respect to rotavirus (12). ACKNOWLEDGMENTS This work was supported by the Division of Advanced Nuclear Systems and Projects, U. S. Department of Energy, Washington, D.C., and the Municipal Environmental Research Laboratory, U. S. Environmental Protection Agency, Cincinnati, Ohio, under interagency agreement E(29-2)-3536/ EPA-AG-D LTERATURE CTED 1. Fenner, F., and D. 0. White Medical virology, 2nd ed. Academic Press, nc., New York. 2. Flewett, T. H., A. S. Bryden, and H. A. Davies Virus particles in gastroenteritis. Lancet ii: Flewett, T. H., A. S. Bryden, H. Davies, G. N. Wood, J. C. Bridger, and J. M. Derrick Relation between viruses from acute gastroenteritis of children and newborn calves. Lancet ii: Kapikian, A. Z., W. L. Cline, C. A. Mebus, R. G. Wyatt, A. R. Kalica, H. D. James, D. H. van Kirk, R. M. Chanock, and H. W. Kim A new complement-fixation test for the human reovirus-like agent of infantile gastroenteritis using the Nebraska calf diarrhrea virus as antigen. Lancet i: Kapikian, A. Z., H. W. Kim, R. G. Wyatt, W. J. Rodriguez, S. Ross, W. L. Cline, R. H. Parrott, and R. M. Chanock Reovirus-like agent in stools: association with infantile diarrhrea and development of serologic tests. Science 185: Martin, M. L., E. L. Palmer, and P. J. Middleton Ultrastructure of infantile gastroenteritis virus. Virology 68: Obejeski, J. F., E. L. Palmer, and N. L. Martin Biochemical characterization of infantile gastroenteritis virus (GV). J. Gen. Virol. 34: Smith, E. M., M. K. Estes, D. Y. Graham, and C. P. Gerba A plaque assay for rotavirus SA-11..J. Gen. Virol. 43: Ward, R. L., and C. S. Ashley Discovery of an agent in wastewater sludge that redtuces the heat required to inactivate reovirus. Appl. Environ. Microbiol. 34: Ward, R. L., and C. S. Ashley dentification of detergents as components of wastewater sludge that modify the thermal stability of reovirus and enteroviruses. Appl. Environ. Microbiol. 36: Ward, R. L., and C. S. Ashley ph modificationi ot the effects of detergents onl the stability ot enteric viruses. Appl. Environ. Microhiol. 38: Ward, R. L., and C. S. Ashley Comparative study on the mechanisms of rotavirus inactivation by sodiun dodecyl sulfate and ethylenediaminetetraacetate. Appl. Environ. Microbiol. 39: