Survival of the enteric adenoviruses 40 and 41 in tap, sea, and waste water

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Pergamon

0043-1354(95)00070-4

War. Res. Vol. 29, No. 11, pp. 2548 2553, 1995 Copyright ~ 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0043-1354/95 $9.50 + 0.00

S U R V I V A L OF THE E N T E R I C A D E N O V I R U S E S 40 A N D 41 IN TAP, SEA, AND WASTE WATER C A R L O S E. E N R I Q U E Z ~*, C H R I S T O N J. H U R S T 3@ and C H A R L E S P. G E R B A 1"2® ~Department of Microbiology and Immunology, and -'Department of Soil and Water Science, University of Arizona, Tucson, AZ 85721 and 3U.S. Environmental Protection Agency, Cincinnati, OH 45268, U.S.A. (First received September 1994; accepted in revised form February 1995)

Abstract--The enteric adenoviruses types 40 (Ead 40) and 41 (Ead 41) have emerged as a leading cause of viral gastroenteritis in children, second in importance only to the rotaviruses. The role of the enteric adenoviruses as waterborne pathogens has not been evaluated. This study compared the survival of these agents with poliovirus type 1 (polio I) and the hepatitis A virus (HAV) in tap water at 4°C, and at room temperature, with polio 1 in primary and secondary wastewater at 4, and 15°C, and in sea water at 15°C. Assays were conducted at regular intervals by the TCIDs0 method in PLC/PRF/5 cells. The survival of Ead 40 and Ead 41 in primary and secondary wastewater was slightly greater than that of polio 1. However, in tap, and sea water, the enteric adenoviruses were substantially more stable than either polio 1 or HAV. These results suggest that the enteric adenoviruses may survive for prolonged periods in water, representing a potential route of transmission. Key words--survival, tapwater, wastewater, seawater, hepatitis A, poliovirus, adenovirus

INTRODUCTION Diarrheal diseases are the leading cause of childhood morbidity and mortality in developing countries (Uhnoo et al., 1986). It has been estimated (LeBaron et al., 1990) that infectious gastroenteritis in the U.S.A. alone, causes more than 210,000 children of 5 years of age or younger to be hospitalized, at a yearly cost of nearly 1 billion dollars. Throughout the world, 3-5 billion cases of diarrhea occur, with approx. 5-10 million deaths every year (LeBaron et al., 1990). Adenoviruses 40 and 41 (Ead 40 and Ead 41) have been recognized as important etiological agents of gastroenteritis in children (Uhnoo et al., 1986; Cruz et al., 1990), second in importance only to rotavirus. Greater numbers of adenoviruses than enteroviruses have been consistently found in raw sewage around the world (Irving and Smith, 1981; Hurst et al., 1988; Krikelis et al., 1985a, b), and it has been estimated (Hurst et al., 1988) that 80% of infectious adenoviruses shed in the feces, and present in raw sewage may be enteric adenoviruses. No etiologic agents have been identified in half of the reported waterborne disease outbreaks in the U.S.A. (Sobsey et al., 1993). Therefore, in spite of lack of evidence, the possibility of th enteric adenoviruses as the cause of undocumented waterborne disease exists. Furthermore, the transmission of waterborne conjunctivitis *Author to whom all correspondence should be addressed.

by adenovirus types 3 and 4 has been established (Rao and Melnick, 1986; Grabow, 1990). Current priorities in enteric viral research have been identified by The Centers for Disease Control, these include: (1) improving diagnostic capabilities; (2) identifying the etiology of the 50% of gastroenteritis outbreaks that are still of unknown origin; and (3) determining the modes of transmission, and the means to prevent disease (LeBaron et al., 1990). Outbreaks caused by enteric adenovirus have occurred mostly in hospitals and day-care centers, affecting only children (Chiba et al., 1983). The enteric nature of Ead 40 and Ead 41, their presence only in the gastroenteric tract, and their extensive geographical distribution suggest that water may play a role in the transmission of these agents. The study of the enteric adenoviruses in water has been hindered because these agents propagate poorly, or not at all, in conventional human cell lines in which other adenoviruses can be propagated effectively (deJong et al., 1983). However, recently, the successful propagation of both, Ead 40 and Ead 41 in P L C / P R F / 5 human liver cell line was reported (Grabow et al., 1992). The availability of this cell line provides a practical method for the study of these viruses in the aquatic environment. This study compared the survival of Ead 40 and Ead 41 with poliovirus type 1, in tap, sea, and waste water, at 4, 15°C, and room temperature (23:C), and with the hepatitis A virus (HAV) in tap water, at 4:C, and room temperature (23 C).

2548

Survival of enteric adenovirus in water MATERIALS AND METHODS Production o f viruses and tissue culture assay

Poliovirus type 1 (strain LSc2ab) (polio 1) was propagated in the Buffalo green monkey (BGM) kidney continuous cell line. The cells were grown and maintained in minimum essential medium (MEM) (Flow Laboratories, McLean, Va) containing 5 and 2% fetal bovine serum (FBS), respectively. Hepatitis A virus (HAV) (strain HMI75), and the enteric adenoviruses types 40 (Ead 40) (strain Dugan) and 41 (Ead 41) (strain TAK) were propagated on human primary liver carcinoma cells (PLC/PRF/5) (Grabow et al., 1983). Cell debris from virus suspensions were removed by low-speed centrifugation (2000g for 10 min), followed by extraction twice with freon (Trichlorotrifluoroethane, Fisher Scientific, Pa). These preparations were frozen at 20':C before use. Virus titration was carried out by the tissue culture infectious dose fifty (TCIDs0) procedure, on PLC/PRF/5 cells in 96-well plates (Becton Dickinson Labware, N.J.), modified from Precious and Russell (1985). Survival is reported as the logloNt/No TCIDs0/ml, which expresses the reduction in viral numbers at each time interval, where N o and N, are the initial and final viral titers respectively. Viral rate of inactivation, correlation coefficient of the data (time vs viral inactivation), and time for 99% virus inactivation (T99) were calculated using linear regression analysis (Yates et al., 1985). The t-test statistic was used to determine significant differences in survivability between viruses at given conditions. Source o f water

Tap water was obtained directly from the tap at the University of Arizona, Tucson. The water is supplied from a well located on the University of Arizona campus. Absence of total and free chlorine in tap water was determined by the N,N-diethyl-p-phenylenediamine (DPD) method (Hach Company, Loveland, Colo.). Settled primary and secondary sewage samples were collected in sterile 1-1 plastic bottles, at the Roger Road wastewater treatment facility in Tucson, Ariz., and sea water was obtained from the coastal area of California, near Los Angeles. Chemical analysis of tap, sea, and waste water were adapted from Standard Methods Jbr the Examination of Water and Wastewater, and are listed in Tables 1 and 2, respectively. A total of 248 samples were examined in this study. Samples of tap and sea water, and primary and secondary wastewater effluents, were kept in a BOD incubator (LabLine Instruments, Melrose Park, I11.), at either 4 or 15°C, and individually seeded, with approx. 1 × 104 TCIDs0/ml of Ead 40, Ead 41, and polio 1. Samples of tap water, kept at room temperature, were additionally seeded with approx. 1 × 104 TC1Ds0/ml of HAV. Samples were aliquoted in 1.5-ml polypropylene tubes, incubated from 50 to 60 days, at the indicated temperatures, and withdrawn for assay at regular intervals. RESULTS Overall, the enteric adenoviruses were more stable t h a n either polio l or HAV. In tap water, at 4°C Ead 41 lost less t h a n 0.5 logt~, and Ead 40 lost nearly one logl0 during a 55-day period [Fig. l(a)], while the titre of H A V and polio 1 decreased approx, i.6 and 2.7 log~0, respectively [Fig. l(a)]. The predicted time for 99% inactivation of these viruses varied with virus type a n d experimental conditions. The 7"99values for polio 1, HAV, Ead 40, a n d Ead 41, u n d e r these conditions, were 41, 56, 92 and 304 days, respectively (Table 3). U n d e r these conditions, t-test statistic analysis of the data did not show significant differWR 29 [ I - - H

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Table I. Tap water characteristics Parameter Value Free chlorine (mg/I) 0 Total chlorine (mg/l) 0 pH 7.7 Alkalinity (mg/I) 90 Total hardness (mg/l) 134 Ca hardness (mg/l) 132 Mg hardness (mg/l) 2 Nitrogen (nitrate) (mg/I) 3.5 Nitrogen (ammonia) (mg/l) 0.05 Turbidity (NTU) 0.35

ences between the survival o f polio i a n d HAV; H A V a n d Ead 40; a n d Ead 40 and Ead 41. However, a significant difference in the survivability was observed between polio 1 a n d Ead 40 (0.05 significance); polio 1 a n d Ead 41 (0.01 significance); and H A V a n d Ead 41 (0.025 significance). In tap water at 15°C, the titer of polio 1 declined nearly 3 lOgl0 after 42 days, whereas, by day 60, the titers of b o t h Ead 40 a n d Ead 41 were only reduced 1.4 a n d 1.21og~0, respectively [Fig. I(b)]. The estim a t e d T99 value for polio I was of 24 days, whereas for Ead 40 a n d Ead 41 was of 87 a n d 124, respectively (Table 3). Using t-test analysis, the difference in survival between polio 1 and b o t h Ead 40 a n d Ead 41 was significant at the 0.05 level. In tap water at r o o m temperature, Ead 40 a n d Ead 41 also were more stable t h a n b o t h polio 1 and HAV. The titers of Ead 40 a n d Ead 41, after 55 days, decreased nearly 2 logl0 [Fig. l(c)]. Polio 1 and HAV, on the o t h e r hand, lost approx. 4, a n d 3.5 log~0 after 30 and 50 days, respectively [Fig. l(c)]. The predicted n u m b e r of days required for 9 9 % viral inactivation at r o o m temperature in tap water were 60 a n d 84 for Ead 40 and Ead 41, respectively, whereas the 7"99 value for H A V was 27 a n d t h a t for polio 1 was 11 (Table 3). Significant differences in the viral survivability in tap water held at room t e m p e r a t u r e were detected, using t-test analysis, between polio 1 and Ead 40 and Ead 41 (significance of 0.05 a n d 0.01, respectively), but not between H A V a n d Ead 40 or Ead 41. In sewage effluents, the survival of the Ead 40 a n d Ead 41 was slightly superior to that of polio 1. However, no significant difference was found using t-test analysis. In primary wastewater, after 50 days at 4°C, the titer o f Ead 41 diminished by a b o u t 2 log~0, t h a t of Ead 40 nearly 2.5 log~0, a n d that of polio 1 2.2 logt0 [Fig. 2(d)]. In primary effluent after 60 days at 15°C, b o t h enteric adenoviruses survived

Table 2. Primary and secondary sewage effluents, and characteristics Primary Secondary Parameter effluent effluent BOD5 (mg/l) 195 16 pH 8.0 7.7 Turbidity (NTU) 40 18 Salinity (ppt) ND ND ND = not determined.

sea water Sea water ND 8.1 3.4 35

2550

Carlos E. Enriquez et al. (a) Tap water at 4°C

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slightly longer than polio l, with a decrease of about 2.6 and 2.8 logj0 for Ead 41 and Ead 40, respectively [Fig. 2(a)]. Under similar conditions, the titer of polio 1 decreased by approx. 2.7 log~0, but after 42 days [Fig. 2(a)]. The estimated T99 for polio 1, Ead 40 and Ead 41, held at 4 C in primary sewage was of 36, 44, and 48 days respectively. At 15°C, slightly shorter T99 values were calculated, with 28 days for polio 1, 40 for Ead 40, and 43 for Ead 41 (Table 3). In secondary sewage held at 4°C for 50 days, the enteric adenoviruses 40 and 41 showed a similar pattern of inactivation as that of polio 1, with a titer reduction of 1.9, 2.2 and 2.51og10, respectively [Fig. 2(b)]. However, at 15°C after 60 days, Ead 40 and Ead 41 survived longer than polio 1, with a titer reduction of 2.4 and 2.91og10, respectively, and 3.2 log~0 after 35 days for polio 1 [Fig. 2(c)]. The predicted T99 values for polio 1, and Ead 41 incubated at 4°C in secondary sewage were very similar, 49 and 47 days respectively, while Ead 40 showed a 7"99value of 58 days under the same conditions (Table 3). At 15°C, however, polio 1 showed a T99 value of 19, which, was less than half of those of Ead 40 and Ead 41 (43 and 45, respectively) (Table 3). Survival of polio 1 in sea water kept at 1 5 C was shorter than survival of Ead 40 and Ead 41. The polio 1 titer was reduced by 3 log~0 after 28 days, whereas, after 60 days, the titer of Ead 40 and Ead 41 dropped

only 1.4 and 1.6 log10, respectively [Fig. l(d)]. In sea water under the described conditions, the T99 values of 85 days for Ead 41, and 77 for Ead 40 were nearly 4 times larger than the 7"99value of 18 days for polio 1 (Table 3). Analysis by t-test statistic showed a significant survivability difference between polio 1, and Ead 40 or Ead 41 (significance 0.05). All experimental treatments for viral survival showed a significant correlation between inactivation rate, and incubation time, with P values of 0.002 or lower (Table 3). DISCUSSION

Although, the enteric adenoviruses are a major cause of viral gastroenteritis in children (Unhoo et al., 1986), information on their stability in water is not available. This study demonstrated that these agents are more stable than polio 1 in tap water, sewage, and sea water, and more stable than H A V in tap water. Our results are in agreement with those of Bagdasar and Abieva (1971), who reported that adenovirus type 5 survived longer than both poliovirus 1 and echovirus 7 in tap water, at either 4 or 18 C , and with those of Sobsey et al. (1986), who found that HAV in groundwater was more stable than polio 1 at different temperatures. In this study, H A V was more resistant than polio I at both 4:C [Fig. l(a)], and

Survival of enteric adenovirus in water room temperature [Fig. l(c)]. However, it was less stable than the enteric adenoviruses in tap water at either temperature [Fig. 1(a, c)]. It has been suggested (Sobsey et al., 1986) that hepatitis-waterborne outbreaks caused by HAV may be related to the higher survivability of this agent in water. The longer survival of the enteric adenoviruses in tap and sea water in comparison to HAV and polio 1, observed in this study, suggests that waterborne transmission of enteric adenoviruses is a possibility. As with other viruses (O'Brien and Newman, 1977; Yates et al., 1985), temperature seems to play a key role in the survival of enteric adenoviruses in water. In particular, in the case of tap water held at 4°C [Fig. l(a)]. After 55 days, the Ead 41 and Ead 40 lost nearly 0.5 and 1.0 log~0, respectively, with an estimated T99 value of 304 days for Ead 41, which was nearly 8 times larger than that of polio 1, and a T99 value of 92 days for Ead 40, which was more than twice the T99 value of polio 1 (Table 3). In contrast, in tap water at room temperature during the same period, these agents lost almost 2 log~0 [Fig. l(c)]. Some evidence of the higher thermal stability of the enteric adenoviruses in comparison to polio 1 was obtained in an experiment in which both, Ead 40, and polio 1 were held at 50 and 65°C in phosphate buffered saline. After incubation at 50°C for 6 min,

Table 3. Viral inactivation rate, and T99 values under different incubation media and temperatures

Virus type

Inactivation rate (Iogt0/d)

T a p water at 4 C Polio I 0.04571 HAV -0.03246 Adeno 40 -0.02125 Adeno 41 -0.00653 T a p water at 15"C Polio 1 0.06209 Adeno 40 -0.02412 Adeno 41 -0.01578 T a p water at 23"C Polio 1 -0.12461 HAV -0.07376 Adeno 40 0.03080 Adeno 41 -0.02639 Secondary sewage effluent at 4 ' C Polio 1 0.04379 Adeno 40 -0.03636 Adeno 41 -0.04425 Secondary sewage effluent at 15 C Polio 1 -0.08143 Adeno 40 -0.04633 Adeno 41 -0.03784 Primary sewage effluent at 4 C Polio I -0.04901 Adeno 40 -0.04705 Adeno 41 0.04483 P r i m a r y sewage effluent at 15 C Polio 1 -0.05908 Adeno 40 -0.04674 Adeno 41 -0.04520 Sea water at 1 5 C Polio 1 -0.11106 Adeno 40 -0.02570 Adeno 41 -0.02528

r

p

T99

-0.971 -0.944 -0.943 -0.844

~<0.0001 ~<0.0001 ~<0.0001 ~<0.0001

41 56 92 304

-0.949 0.988 -0.911

0.001 ~<0.0001 0.002

24 87 124

-0.955 0.985 -0.900 -0.873

0.001 ~<0.0001 ~<0.0001 ~<0.0001

11 27 60 84

-0.938 -0.974 0.964

0.001 ~<0.0001 ~<0.0001

49 58 47

-0.965 -0.983 0.958

0.0021 ~<0.0001 ~<0.0001

19 43 45

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~<0.0001 ~<0.0001 ~<0.0001

36 44 48

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~<0.0001 ~<0.0001 ~<0.0001

28 40 43

-0.996 0.994 -0.975

~<0.0001 ~<0.0001 ~<0.0001

18 77 85

r = correlation coeffcient, p = level o f significance, T99 = time in days for 9 9 % inactivation o f the original titer.

2551

polio 1 lost 0.88 log10, whereas Ead 40 lost less than 0.2 log~0. Similarly, at 65°C, polio 1 lost more than 2.5 log~0 in 30 s, while Ead 40 lost only 1 log~0 after the same incubation time (data not shown). The fact that temperature plays an important role in viral survival is further supported by LaBelle and Gerba (1982), who reported that the titer of polio 1 in sea water at 9°C dropped only 2 log~0 in 35 days, while in our experiment at 15°C in sea water we observed a similar reduction, but in approximately half the time [Fig. l(d)]. In contrast, at a higher temperature, Loh et al. (1979), and Fujioka et al. (1980), showed that incubation of polio 1 in sea water at 24°C, resulted in a rapid viral inactivation with nearly 3 log~0 in 4 days. Similarly, in situ and laboratory inactivation experiments of polio 1 in sea water at 20°C (Jofre et al., 1986) have resulted in fast inactivation rates of 3.5 and 4 log~0 in 5 days, respectively. Although, viral survival studies are difficult to compare, as they are carried out under different conditions, Callahan et al. (1994) demonstrated that polio 1 inactivation in sea water was very similar using three geographically diverse types of sea water. This rapid inactivation suggests that not only temperature, but other factors are associated with viral survival in ocean waters. The poor survival of polio 1 in sea water could have been related to salinity, which has been negatively associated with viral survival in sea water, due to viral aggregation, and marine microflora (Gerba and Schaiberger 1975; Fujioka et al., 1980; La Belle and Gerba, 1982). The enteric adenoviruses did not survive significantly longer than polio 1 in primary and secondary sewage at either 4 or 15°C, with an inactivation rate of approx. 2.5 log~0 in 50 days [Fig. 2(a~t)]. This result was unexpected, as organic matter present in water samples is known to protect enteroviruses from inactivation (La Belle and Gerba, 1982). The increased survivability of the enteric adenoviruses in tap and sea water, and the unexpected faster inactivation in sewage, may indicate that these viruses are inactivated by different mechanisms than those affecting the enteroviruses. The increased resistance showed by enteric adenoviruses may be associated to the double stranded nature of their DNA, which if damaged, may be repaired by the host cell DNA-repair mechanisms, which in human cells, in addition to repairing pyrimidine dimers, can also repair a wide range of DNA damages (Bernstein and Bernstein, 1991). In addition, both adenovirus DNA strands may serve as template for replication (Kelly, 1984). Thus, if one strand is damaged by environmental factors, the other may still serve as template for replication of progeny DNA. This mechanism would not be effective on ssRNA-genome viruses like polio 1 or HAV. It has been suggested (Enriquez et al., 1993), that nucleic acids from DNA viruses would persist longer in the

2552

Carlos E. Enriquez et al.

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Days Fig. 2. Survival poliovirus l (O), enteric adenovirus 40 (*), and enteric adenovirus 41 ([B). aquatic environment, as indigenous D N A a s e s require cofactors to be active, and are denatured by temperature more readily than RNAases. It could be speculated then, that the longer survival o f the enteric adenoviruses in tap and sea water might have been associated to D N A damage that could have been repaired by the host cell, while the faster inactivation in sewage might have resulted from protein capsid damage, rendering the virions unable to enter the host cell. This hypothesis could be partially tested by conducting inactivation experiments as described, but assaying the samples on both normal cells, and cells lacking the ability to repair D N A . From the results obtained in this study, it can be concluded that the enteric adenoviruses are more stable in water than polio 1. As this agent has been used extensively as a model for viral inactivation in water (Sobsey et al., 1986), its use to assess enteric adenovirus survival in water, therefore, would not be appropriate. The greater stability o f the enteric adenovirus, in tap water, in comparison to H A V is o f concern, as the waterborne transmission o f this agent has been d o c u m e n t e d (Sobsey et al., 1993). In order to assess the public health risk associated with the exposure to enteric adenoviruses in water, it would be necessary to know the occurrence of these agents in the aquatic environment. Unfortunately, this data is not available. Therefore, future research should be

implemented to determine the occurrence o f enteric adenoviruses in water. REFERENCES

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La Belle R. and Gerba C. P. (1982) Investigations into the protective effect of estuarine sediment on virus survival. Wat. res. 16, 469-478. LeBaron C. W., Furutan N. P., Lew J. F., Allen J. R., Gouvea V., Moe C. and Monroe S. S. (1990) Viral agents of gastroenteritis. M M W R 39, 1-24. Loh P. C., Fujioka R. S. and Lau L. S. (1979) Survival and dissemination of human enteric viruses in oceans waters receiving sewage in Hawaii. Wat. Air, Soil Pollut. 12, 197-217. O'Brien R. T. and Newman J. S. (1977) Inactivation of polioviruses and coxsackieviruses in surface water. Appl. environ. Microbiol. 33, 334-340. Precious B. and Russell W. C. (1985) Growth, Purification and Titration o f Adenoviruses. In Virology a Practical Approach (Edited by Mahy B. W.). IRL Press, Washington, D.C. Rao V. C. and Melnick J. L. (1986) Environmental Virology. Am. Soc. Microbiology, Washington, D.C. Sobsey M. D., Shields P. A., Hauchman F. S., Hazard R. L. and Caton L. W. (1986) Survival and transport of hepatitis A virus in soils, groundwater and wastewater. Wat. Sci. Technol. 18, 97-106. Sobsey M. D., Dufour A. P., Gerba C. P., LeChevallier M. W. and Payment P. (1993) Using a conceptual framework for assessing risks to health from microbes in drinking water. J. Am. Wat. Wks Assoc. Sobsey M. D., Shields P. A., Hauchman F. H., Hazard R. L. and Caton W. (1986) Survival and transport of hepatitis A virus in soils, groundwater and wastewater. Wat. Sci. 18, 97 106. Uhnoo I., Wadell G., Svensson L., Olding-Stenkvist E. and Molby R. (1986) Aetiology and epidemiology of acute gastroenteritis in swedish children. J. Infect. 13, 73 89. Yates M. V., Gerba C. P. and Kelly L. M. (1985) Virus persistence in groundwater. Appl. environ. Microbiol. 49, 778-781.