Stability of Simian Rotavirus in Fresh and Estuarine Water

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 198, p /8/1-1/5$2./ Vol. 39, No. 1 Stability of Simian Rotavirus in Fresh and Estuarine Water CHRISTON J. HURST AND CHARLES P. GERBA* Department of Virology and Epidemiology, Baylor College ofmedicine, Houston, Texas 773 The rates of inactivation of poliovirus 1, echovirus 7, coxsackievirus B3, and simian rotavirus SAil were compared in polluted and nonpolluted fresh and estuarine water samples. The study was done in two parts, comparing virus survival in samples taken 1 year apart from the same sites. The survival studies were perforned at 2C and at the natural ph of the water samples. In the first part of the study, the time required for a 3-logio reduction in the initial virus titers was 2 to 3 days in the estuarine water samples and varied from 3 to >14 days in the freshwater samples. In the second part of the study, no clear distinction was found between survival of viruses in freshwater samples and survival in estuarine water samples. The time required for a 3-logio reduction in the initial virus titers in the second part of the study varied from 12 to >14 days. This indicates that there is a nonseasonal change in factors in the water which affect virus survival. In this study SA11 survival time (used as a model for human virus) was well within the range exhibited by the enteroviruses, indicating that it also is environmentally stable in natural waters. It has been known for some time that members of the picornavirus family can be isolated from sewage-contaminated fresh and estuarine water (1, 17). Such waters may eventually be used for consumption and recreational purposes, thus posing a public health threat (7, 9, 15). Consumption of raw shellfish from sewage-contaminated water has been implicated as the source of outbreaks of hepatitis A (7, 15). Two excellent reviews concerning the survival of viruses in ground and surface water'are those of Akin and co-workers (1, 2). Acute infantile gastroenteritis is one of the most common causes of childhood disease throughout the world and is a leading cause of mortality in many underdeveloped countries (1). Recent studies have indicated that rotaviruses may be the causative agents in nearly onehalf of all hospitalized cases of acute diarrheal illnesses in infants and young children (5, 8). Rotaviruses have also been implicated in epidemics of acute gastroenteritis in adults (14) and have been suspected in a recent waterborne outbreak (12). Patients who are ill with rotavirus gastroenteritis excrete the virus at levels up to 19 particles per g of feces (1). Therefore, the probability that infectious rotavirus may find its way into sewage treatment plants exists. The objective of this study was to determine whether rotaviruses were able to survive long enough in surface waters to pose a public health problem if they escape inactivation during the sewage treatment process and are discharged into effluent receiving waters. MATERIALS AND METHODS Virus and viral assays. Plaque-purified strains of echovirus 7 strain Wallace, coxsackievirus B3 strain Nancy, poliovirus 1 strain LSc, and simian rotavirus SAll were used. The enterovirus stocks were passaged and assayed in BGM cells, a continuous monkey kidney cell line. Enterovirus assays were done by the plaque-forming unit method, as described previously (13). Simian rotavirus SAll was grown and assayed by a technique developed in our laboratory (18). Rotavirus for the first experiment was grown and assayed in LLC-MK2, a continuous rhesus monkey kidney cell line. Rotavirus for the second experiment was grown and assayed in MA14 cells, a continuous line of fetal rhesus monkey kidney. Basically, the SAll plaque assay method is as follows. Flint glass bottles (1 ounce [3 ml]) were seeded with complete Eagle minimal essential medium containing MK2 or MA14 cells. The bottles were incubated at 37 C until the cells produced a confluent monolayer. The medium was then poured off the monolayer, the monolayer was washed with serum-free medium, and each bottle was inoculated with.1 ml of virus-containing sample. After inoculation, the virus was allowed to adsorb to the cell monolayer for 1 h at 37 C. The cell monolayer in each bottle was then overlaid with 5 ml of complete Eagle medium (without phenol red) containing 2 jig of neutral red per ml, 1.5% agar, 1 U of penicillin per ml, 1,g of streptomycin per ml,.3% sodium bicarbonate, 3,ug of glutamine per ml, 1:6 Oxoid buffered pancreatin, and 1 yg of diethylaminoethyl-dextran per ml. The inoculated cell cultures were then incu-

2 2 HURST AND GERBA bated at 37 C for 2 weeks; clear areas (plaques) appearing on the cell layer during this time were counted. The culture system for the two experiments was changed because interim studies by others indicated that MA14 cells may be more sensitive. All cell culture lines were grown in Eagle minimal essential medium containing glutamine and 1% fetal calf serum. If necessary, enterovirus samples were diluted in tris(hydroxymethyl)aminomethane buffer containing (per liter) 8 g of NaCl,.38 g of KCI,.1 g of Na2HPO4, 1 g of dextrose,.1 g of MgCl2,.1 g of CaCl2, and.2 g of phenol red; this buffer also contained 1 U of penicillin per ml and 1l,g of streptomycin per ml. Samples containing rotavirus were diluted in a 5% tryptose phosphate broth solution (BBL Microbiology Systems, Cockeysville, Md.). The salinity of the water samples did not appreciably affect the plating efficiency of the viral assays. Water samples. Water samples for use in the survival experiments were collected from several estuarine sites on the east coast of Texas. The water at these sites was receiving sewage discharges. Water was also collected from a nonpolluted (as determined by the presence of fecal coliforms), freshwater, city park pond, as well as from a heavily sewage-polluted Houston stream which was receiving effluent from several sewage treatment plants. Salinity was determined by using an AO T/C refractometer (AO Instruments Corp., Buffalo, N.Y.). All water samples for a given experiment were collected on the same day within a few hours of the start of the experiment. The two experiments were begun exactly 1 calendar year apart to avoid any possible seasonal variations in factors affecting virus survival in the samples. The water was used as collected for the survival experiments, with no filtration or other pretreatment. Procedure for measuring survival. Each of the water samples was divided into 1-ml portions that were placed in sterile flint glass bottles. The initial titer of virus in each bottle was approximately 2 x 14 plaque-forming units of poliovirus, echovirus, coxsackievirus, or rotavirus per ml. The bottles were shaken vigorously for 5 s, after which a 1-ml sample was withdrawn from each bottle. The samples were immediately diluted in the appropriate solution and stored at -2 C before being assayed for infectivity. The bottles were maintained loosely capped at 2 C. All bottles were sampled on the initial day and on days 1 through 6, 8, 1, 12, 14, and 21. Total sample volumes of 1 ml were assayed to determine the final endpoints for the survival curves. For all graphs, virus survival was plotted as the survival ratio value (logio Nt/No, where Nt is the virus titer at elapsed time t in days and No is the virus titer at the beginning of the experiment, i.e. zero time) versus elapsed time in days since the beginning of the experiment. In the figures, no virus titer values are listed beyond the time at which a 3-log1o reduction in titer had occurred. APPL. ENVIRON. MICROBIOL. RESULTS The results of the first survival experiment are presented in Fig. 1 and 2. Figure 1 shows that for each type of water examined, all viruses were inactivated at roughly similar rates. Reductions of 99.9% (3 loglo) in viral titer occurred much more rapidly in the estuarine water samples (2 to 3 days) than in the freshwater samples (3 to >14 days). Figure 2 shows that, with the exception of echovirus 7, the survival times of each virus were nearly the same in freshwater samples from polluted and nonpolluted sources. Echovirus 7 was very different in this respect, surviving more than twice as long in the polluted freshwater as in the nonpolluted freshwater. The data from this first experiment also indicated that, at least within the estuarine salinity ranges which were examined, the rate of viral inactivation appeared to be unrelated to salt concentration. In general, echovirus 7 survived for a slightly shorter time in the estuarine water than did the other viruses, and coxsackievirus B3 survived slightly longer. In all cases, the survival of SAll was well within the range exhibited by the other enteric viruses examined. The results of the second experiment (Fig. 3) indicated that survival rates of different virus types, in this case SAll and poliovirus 1 as a representative enterovirus type, were not always parallel in any given water sample. In this experiment, different survival rates were again found for rotavirus in water samples from the various sites, whereas the inactivation curves for poliovirus were similar in all of the water samples. The time required for a 3-logi reduction in virus titers in the second experiment varied from 12 to >14 days. DISCUSSION A number of studies concerning the survival of enteric viruses in water of either fresh or saline nature have been published. The rates of inactivation and survival statistics that have been presented for enteroviruses have varied widely, undoubtedly due to differences in experimental conditions and variations in the source of water employed. No infornation is available at present concerning the survival of rotaviruses in water. Simian rotavirus SAll was used as a model for the rotavirus group, since techniques are not yet available for the growth and quantitation of human rotavirus by conventional tissue culture methodology. However, all rotaviruses are structurally identical and are closely related antigenically (1). The objective of the experiments presented here was to compare simultaneously the rates of inactivation and survival times for SAl rotavirus and three enteroviruses in water of both fresh and saline origin. We felt that the results for different water samples would be directly comparable only if the experiments were done simultaneously, due to daily differences in water

3 VOL. 39, 198 VIRUS SURVIVAL IN FRESH AND ESTUARINE WATER 3 B R Das C D FIG. 1. Virus inactivation rates with respect to water type. (A) Nonpolluted freshwater. (B) Heavilypolluted freshwater. (C) Estuarine water (salinity, 12g/kg). (D) Estuarine water (salinity, 21 g/kg). (E) Estuarine water (salinity, 28 g/kg). Symbols: E, poliovirus 1; A, echovirus 7;, coxsackievirus B3; O, SAII. conditions at the sampling sites and the necessity to use water samples that had not been stored for any extended period of time before use. Long-term storage might result in modifications of ph, microbial populations, or organic content. Some similarities do exist between our results for virus survival in freshwater and those of Clarke et al. (4), as reviewed by Akin and coworkers (1). Both studies demonstrated that the survival time of poliovirus 1 at 2C was nearly the same in heavily sewage-polluted water and in either minimally polluted or nonpolluted water. The data from both studies also indicated that at 2C echovirus 7 survived more than twice as long in heavily polluted water than it did in slightly polluted or nonpolluted water. In general, our results indicated that viral inactivation in estuarine water was independent of salinity. The same observation has been made previously for the same strain of poliovirus (3), for enteroviruses other than the ones examined here (11), and for bacteriophage T7 (16). For the SAll rotavirus used in these experiments, the similarity of virus survival rates in waters of different salinities did not always hold true. For this reason we concluded that the factors which affect survival of viruses in different waters do change, at least with respect to those affecting survival of rotavirus. A previous study by Akin et al. (1) also indicated a difference in poliovirus 1 inactivation when estuarine water samples from the same site collected at different times were used. Previous work in this laboratory has shown that rotaviruses bind less efficiently to activated sludge solids than do enteroviruses (6). Thus, sewage treatment processes that are efficient in the removal of enteroviruses may not be equally effective for rotaviruses, resulting in the possible discharge of infectious rotavirus into effluent receiving water. To examine the extent of this problem, we are currently developing methods for the concentration and detection of rotaviruses in water and wastewater. The results of the present study indicate that rotaviruses can

4 4 HURST AND GERBA APPL. ENVIRON. MICROBIOL. z U O C A L I FIG. 2. Virus inactivation with respect to virus type: experiment 1. (A) Poliovirus 1. (B) SAlI. (C) Coxsackievirus B3. (D) Echovirus 7. Symbols: E, nonpolluted freshwater; U, heavily polluted freshwater;, estuarine water (salinity, 12 g/kg); A, estuarine water (salinity, 21 g/kg); x, estuarine water (salinity, 28 g/kg). survive long enough in natural waters to be ~A transmitted by this route, and polluted sources of drinking and recreational waters could act as vectors in facilitating the spread of rotavirus -2 from one community to others. Of course, it would be necessary to validate this assumption if methodology for the study of human rotavi- -3 ruses becomes available. ACKNOWLEDGMENTS -4 ~This work was sponsored in part by the Texas A & M ffi ".s University Sea Grant College Program, which is supported by the National Oceanic and Atmospheric Administration Office of Sea Grant, Department of Commerce, under grant LITERATURE CITED \B1. 13 Akin, E. W., W. H. Benton, and W. F. Hill, Jr \ \Enteric viruses in ground and surface waters: a review of their occurrence and survival, p In V. Griffin and V. Snoeyink (ed.), Virus and water quality: occur- 2- rence and control. University of Illinois Press, Urbana- \ tchampaign. 2. Akin, E. W., W. F. Hill, and N. A. Clarke Mortality of enteric viruses in marine and other waters, FIG. 3. Virus inactivation with respect to virus -4 - type: experiment 2. (A) Poliovirus 1. (B) SAlI. Sym- I,, bols: U, heavily polluted freshwater; A, estuarine water (salinity, 26 g/kg); x, estuarine water (salinity, 3 g/kg).

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