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Survival of infectious Poliovirus-1 in river water compared to the persistence of somatic coliphages, thermotolerant coliforms and Poliovirus-1 genome
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Water Research 38 (2004) 2927–2933
Survival of infectious Poliovirus-1 in river water compared to the persistence of somatic coliphages, thermotolerant coliforms and Poliovirus-1 genome S. Skraber, B. Gassilloud, L. Schwartzbrod, C. Gantzer* LCPME–UMR 7564 CNRS-UHP, Faculte´ de Pharmacie, 5 rue Albert Lebrun, B.P. 403, 54001 Nancy, France Received 1 September 2003; received in revised form 8 March 2004; accepted 26 March 2004
Abstract The microbiological quality of water is currently assessed by search for fecal bacteria indicators. There is, however, a body of knowledge demonstrating that bacterial indicators are less resistant to environmental factors than human pathogenic viruses and therefore underestimate the viral risk. As river water is often used as a resource for drinking water production, it is particularly important to obtain a valid estimation of the health hazard, including specific viral risk. This work was conducted to compare the survival of infectious Poliovirus-1 used as a pathogenic virus model to the persistence of, on the one hand, thermotolerant coliforms commonly used as indicators and on the other hand, to somatic coliphages and Poliovirus-1 genome considered as potential indicators. We studied the behavior of infectious Poliovirus-1 and the three (potential) indicators of viral contamination in river water at three different temperatures (4 C,18 C and 25 C). This experiment was performed twice with river water sampled at two different periods, once in winter and once in summer. Our results showed that the survival of thermotolerant coliforms can be 1.5-fold lower than infectious Poliovirus-1. In contrast, under all our experimental conditions, somatic coliphages and Poliovirus-1 genome persisted longer than infectious Poliovirus-1, surviving, respectively, 2–6-fold and about 2-fold longer than infectious Poliovirus-1. According to our results exclusively based on survival capacity, somatic coliphages and viral genome, unlike thermotolerant coliforms appear to be better indicators of viral contamination in river water. Moreover, the disappearance of viral genome is well-correlated to that one of infectious virus irrespective of the conditions tested. r 2004 Elsevier Ltd. All rights reserved. Keywords: River water; Viral indicator; Somatic coliphages; Thermotolerant coliforms; Virus genome; Poliovirus-1; Persistence; Survival
1. Introduction The microbiological quality of rivers used as a drinking water resource is currently evaluated by measuring the density of bacterial indicators (thermo*Corresponding author. LCPME, Faculte´ de Pharmacie, 5 rue Albert Lebrun, B.P. 403, 54001 Nancy, France. Tel.: +3303-83-68-22-91; fax: +33-03-83-68-23-01. E-mail address: [email protected] (C. Gantzer).
tolerant coliforms, Enterococci, spores of sulfite-reducing anaerobes). These fecal indicators should estimate the risk of the presence of enteric pathogens discharged into the river from a wastewater treatment plant upstream from the drinking water source. This objective can be achieved only if the indicator organisms behave in the same way as pathogens in the water flow of the river or during the process of producing drinking water. It has however been largely demonstrated that this prerequisite is not met when bacteria are used as indicators of enteric viruses pathogenic for humans like
0043-1354/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2004.03.041
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Enterovirus, hepatitis virus and viruses that cause gastroenteritis [1–4]. In a recent publication [5], we demonstrated that in the Moselle river, thermotolerant coliforms and Enterococci decrease much more rapidly than fecal viruses represented by somatic coliphages or F-specific phages especially when temperature is high and flow is low (summer period). This observation demonstrates that viral pollution can be underestimated in river water before collection for drinking water production. Different solutions can be proposed to overcome this problem. As suggested by several authors [6,7] direct detection of pathogenic Enterovirus using cell culture might be a possible solution. The fact that this method only detects infectious viruses is an advantage but the delay to results is too long (1–3 weeks) and certain viruses which may be present (Norovirus) would go undetected. The detection of viral genome by reverse transcription polymerase chain reaction (RT-PCR) is a very rapid, sensitive and specific technique. Several authors [8,9] have suggested applying this method, which may detect all viral serotypes, using the viral genome as an indicator of viral contamination. However, the presence of viral genome is not proof of the presence of infectious virus, making interpretation hazardous. In some cases, viral genome can persist much longer than the infectious virus [10–12]. The usefulness of viral genome detection therefore depends on the time interval between loss of infectivity and genome degradation. Fecal bacteriophages are also proposed as indicators of viral contamination. The most frequently studied are somatic coliphages [13–16], F-specific phages [14,17–19] and Bacteroides fragilis phages [14,20–22]. These phages are excreted in human stools and may behave like pathogenic viruses in water. Each of these potential indicators has its own advantages and disadvantages and, since the
ideal universal indicator probably does not exist, should be evaluated individually for each application or protection purpose. Based on our previous observations in natural river water conditions [5], we decided to isolate the temperature parameter and study the survival in river water of infectious Poliovirus-1 used as a pathogenic virus model on one hand, and the survival/ persistence of three (potential) indicators of viral contamination (thermotolerant coliforms, somatic coliphages and Poliovirus-1 genome) on the other hand. Indeed, the aim was, in our experimental conditions, to evaluate the value of those three microbiological parameters as indicators of the presence of infectious Poliovirus-1.
2. Materials and methods 2.1. River water River water samples are taken from the Moselle river in which it has been already observed a difference of behavior between bacterial and viral indicators [5]. In the location called Maron, 40 l were drawn from the middle of the river at a depth of 1 m. Samples were drawn twice: one in September 4, 2001 (summer) and one in January10, 2002 (winter). The microbiological and physicochemical properties of the samples are given in Table 1. 2.2. Poliovirus-1 strain A stock suspension of Poliovirus-1 Sabin (Lsc 2ab) was prepared by inoculating a monolayer of BGM cells. After 24–48 h of virus multiplication, the cells were frozen and thawed three times and centrifuged (10,000 g,
Table 1 Microbiological and physicochemical parameters of the river water samples Parameters
Units
Summer sample
Winter sample
Temperature pH Conductivity Turbidity Hydrogenocarbonates Alkalinity Dry matter at 180 C Aluminum Iron Nitrate Orthophosphate Dissolved oxygen KMnO4 oxidability Suspended solids Thermotolerant coliforms Somatic coliphages
C — mS cm1 NTU Mg HCO3 l1 Mg CaO l1 Mg l1 Mg Al l1 mg Fe l1 Mg NO3 l1 Mg PO4 l1 Mg O2 l1 Mg O2 l1 Mg l1 Log CFU 100 ml1 Log PFU 100 ml1
19.2 8.1 469 6.4 63 29 178 0.150 366 4.4 0.16 9.5 6.12 8 3.10 3.29
1.2 7.7 63 3.7 106 49 202 0.085 95 8.5 o0.05 12.6 1.90 o2 3.84 3.52
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60 min.). The titer of the resulting supernatant liquid which constituted the viral stock was about 8.3 log MPNCU ml1. It was kept at –80 C. 2.3. Quantification of infectious virus by cell culture Each sample was treated with 10% antibiotic and antimycotic solution (Sigma A-5955) for 2 h at 37 C. Quantification of infectious Poliovirus-1 was performed in 96-well microplates using BGM cells. Fifty microliters of three successive logarithmic dilutions for each analyzed sample were added to 200 ml of 7.5 104 cells ml1 in minimum essential medium (MEM: Sigma M-5650) solution containing 2% fetal calf serum. Each dilution was seeded in 40 wells. After 6 days incubation at 37 C under 5% CO2 atmosphere, the plates were examined under microscope and the cytopathogenic effect (CPE) was identified. Wells exhibiting a CPE were counted and the most probable number of cytopathogenic units (MPNCU) was calculated [23]. The concentrations were expressed in MPNCU ml1 and the inactivation in function of time was estimated by the calculation of log N/N0 with N and N0 corresponding to the concentrations of infectious Poliovirus-1, respectively, at time t and 0. 2.4. Viral genome quantification (i) Viral RNA extraction: Poliovirus genome was extracted from 140 ml of sample with the Qiamp viral RNA kit (Qiagen, 52904) according to the manufacturer’s instructions. Hence, 60 ml of final extracted RNA solution were obtained. (ii) Primers and probes: Primers and probes used for Poliovirus-1 detection are presented in Table 2. They were designed using Primer Express 1.0 software and the complete sequence of Poliovirus1 (Genbank, access number: NC 002058). (iii) cDNA synthesis: 5 ml of extracted RNA was mixed with 1 m of 10 mM reverse primer (ENT-r) and heated at 95 C for 3 min. 14 m of a mixture containing 4 m of 5 reverse transcription buffer (250 mM Tris-HCl (pH=8.4), 50 mM MgCl2, 350 mM KCl, 15 mM dithiothreitol, 2.5 mM sper-
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midine) (Promega, M515A), 1 ml of 40 U ml1 RNase inhibitor (Promega, N211A), 2 ml of 10 mM of each deoxynucleoside triphosphate (Perkin Elmer N8080260), 1 ml of 8 U ml1 reverse transcriptase (Promega, M510A), 5 ml Dnase/Rnase free water (Sigma W4502) and 1 ml of 1 mg. ml1 T4gene32 (Roche, 972991) were added. Reverse transcription was performed at 42 C for 60 min. Finally, RNA-DNA hybrids were denatured, and reverse transcriptase was inactivated by heating to 95 C for 5 min. The resulting cDNA was then quantified by PCR. (iv) DNA quantification: PCR assay was performed with 5 ml cDNA and 45 ml of the mixture containing 25 ml Taq man universal Master mix (Perkin Elmer, 4304437), 14.5 ml nuclease free water (Sigma W4502), 0.5 mM forward primer, 0.5 mM reverse primer and 0.5 mM probe. Amplification was performed using an ABI Prism 7700 (Perkin Elmer Inc.). The amplification ramp included two hold programs: (i) 2 min at 50 C to activate the uracil N0 -glycosylase followed by (ii) 10 min at 95 C to release the activity of the Hotstart DNA polymerase. Samples were then submitted to 50 cycles (15 s. at 95 C and 1 min at 60 C). Real-time fluorescence measurements were obtained and directly analyzed by the ABI prism 7700 sequence detection system 1.7.1 software (Perkin Elmer). The threshold cycle (Ct) value generated for each sample was calculated by determining the point at which fluorescence exceeded a threshold limit. The viral genome degradation in function of time was estimated by the calculation of log N/N0 with N and N0 corresponding to concentrations of DNA, respectively, at time t and 0. 2.5. Thermotolerant coliforms Thermotolerant coliforms were quantified on m-FC medium after membrane filtration according to norm ISO 9308/1 [24]. The concentrations were expressed in CFU ml1. The inactivation was estimated by the calculation of log N/N0 corresponding to concentrations of thermotolerant coliforms, respectively, at time t and 0.
Table 2 Primers and probe (Eurogentec) Type Primer Primer Probe a
Name ENT-r ENT-f ENT-p
Reaction RT and PCR PCR PCR
f: FAM reporter dye, t: Tamra quencher dye.
Sequence (50 –30 ) 0
Position 0
5 -CAAGATTGGTTCCTGCTTGATCTT-3 50 -AGCATTGTGATCGATGGCAA-30 50 -f-AGATCTTGGATGCCAAAGCGCTCG-t-30 a
5648-5625 5573-5592 5601-5624
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2.6. Somatic coliphages Somatic coliphages were quantified using the bacterial host strain E. coli (WG5) according to standard methods ISO/FDIS 10705-2 [25]. The concentrations were expressed in PFU ml1. The inactivation was estimated by the calculation of log N/N0 corresponding to concentrations of somatic coliphages, respectively, at time t and 0. 2.7. Experimental protocol Concentrations of thermotolerant coliforms and somatic coliphages naturally present in river water samples are given in Table 1. Since the infectious Poliovirus-1 used as a pathogenic virus model is not naturally found in river water, an artificial contamination was performed. River water sample was divided into three equal aliquots of 5 l. One milliliter of the Poliovirus-1 stock suspension was added to each 5-l river water aliquot. The final concentration was 4.7 log MPNCU ml1 in the summer sample and 4.4 log MPNCU ml1 in the winter sample. The high values of concentration (>4 log) allowed to follow up the survival of Poliovirus-1 for several days under constant stirring at 100 rpm. The three 5-l river water samples were held, respectively, at 4 C, 18 C and 25 C. At the same time, three 5-l river water samples were not contaminated with Poliovirus-1 and were held under the same conditions. These samples were used to monitor survival of the bacterial and phagic indicators in river water as a control to verify if the addition of the Poliovirus-1 suspension that may contain nutriments change the behavior of bacteria or viral indicators. Survival was monitored for 2 weeks. This operation was run once in summer and once in winter. The decline in survival was calculated using the ratio log N/N0 where N0 is the concentration at time 0 and N the concentration at time t: As the determination of concentration for all microbiological parameters has been performed at the same time t; each infectious Poliovirus-1 concentration can be associated to an indicator concentration (pairwised parameters). Thus, the decrease of each indicator could be compared with that of infectious Poliovirus-1 for each temperature (4 C, 18 C and 25 C) and for each sample (winter and summer). Linear regression was used to modelize the log reduction of respectively thermotolerant coliforms, somatic coliphages and Poliovirus-1 genome in function of infectious Poliovirus-1 log reduction. The different slopes were compared using Student’s t test.
3. Results As supposed, we observed that when temperature increases, the persistence of thermotolerant coliforms,
somatic coliphages, infectious Poliovirus-1 or its genome decrease. It was also shown that the addition of the Poliovirus-1 suspension had no significant effect on somatic coliphages survival whereas it changed the behavior of thermotolerant coliforms (data not shown). Therefore, we only considered the survival of thermotolerant coliforms and somatic coliphages in river water samples that had not been spiked with Poliovirus-1. To reach our objective, we compared the persistence of thermotolerant coliforms, somatic coliphages and Poliovirus-1 genome to the survival of the infectious Poliovirus-1 in river water sampled in September (summer) and January (winter) and held at 4 C, 18 C and 25 C. For thermotolerant coliforms, when comparing their survival to the persistence of the infectious Poliovirus-1, temperature did not have a significant effect on the slopes of the linear regressions (p-value>0.05). Thus, summer and winter samples were presented with a single regression equation (Fig. 1). In contrast, pooling the data for the three temperatures demonstrated a significant ‘‘sampling date’’ effect (p-valueo0.05). Thus, survival of culturable thermotolerant coliforms in winter water was lower than that of infectious Poliovirus-1 (slope>1) for the temperature range from 4 C to 25 C whereas the opposite is observed in summer water. When comparing the persistence of the viral genome to that one of the infectious Poliovirus-1, a similar pattern was observed. Indeed, temperature did not have a significant effect on the slopes of the linear regressions (p-value>0.05). Summer and winter samples were thus presented with a single regression equation (Fig. 2). As for thermotolerant coliforms, there was a significant difference in behavior depending on the sampling date (p-valueo0.05) although the difference was relatively
Infectious poliovirus (log N/N0) 0
-1
-2
-3
-4
-5
0 Coliforms (log N/No)
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Summer (4, 18 and 25˚C): y= 0.58x + 0.13 2 R = 0.95
-1
-2
-3
-4
Winter (4, 18 and 25˚C): y = 1.30x + 0.05 R2 = 0.91
y=x
Fig. 1. Relation between the log reduction of infectious Poliovirus-1 and the one of thermotolerant coliforms in two river water (summer } and winter n). Each regression takes into account the results obtained at 4 (white), 18 (gray) and 25 C (black) (ntotal ¼ 49).
ARTICLE IN PRESS S. Skraber et al. / Water Research 38 (2004) 2927–2933 Infectious poliovirus (log N/N0)
Poliovirus genome (log N/No)
0
-1
-2
-3
-4
-5
0 Winter (4, 18 and 25˚C): y = 0.56 x - 0.02 R2 = 0.97
-1
-2
Summer (4, 18 and 25˚C): y = 0.71 x +0.08 R2 = 0.97 y=x
Fig. 2. Relation between the log reduction of infectious Poliovirus-1 and the one of Poliovirus-1 genome in two river water (summer } and winter n). Each regression takes into account the results obtained at 4 (white), 18 (gray) and 25 C (black) (ntotal ¼ 49). Infectious poliovirus (log N/N0)
-1
-2
-3
-4
-5
Coliphages (log N/No)
0 Summer (4 and 18˚C): y = 0.17x + 0.21 R2 = 0.69
-1 Winter (4 and 18˚C): y = 0.41x + 0.07 R2 = 0.93
-2
comparisons showed a significant sample date effect (pvalueo0.05). The survival of somatic coliphages, in our experimental conditions, was always 2- to over 6-fold higher than that of infectious Poliovirus-1 (slopeo1).
4. Discussion
-3
0
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y=x
-3
Fig. 3. Relation between the log reduction of infectious Poliovirus-1 and the one of somatic coliphages in two river water (summer } and winter n). Each regression takes into account the results obtained at 4 (white) and 18 C (gray) (ntotal ¼ 35). Results obtained at 25 C (black) are indicated (n ¼ 15) but were analyzed separately.
small (slope in summer water=0.71 versus in winter water 0.56). Persistence of Poliovirus-1 genome was always greater than that of infectious Poliovirus-1 (slopeso1). Finally, when comparing the survival of somatic coliphages to that of the infectious Poliovirus-1, analysis of the linear regressions for the three temperatures demonstrated a significant difference in winter water. There was no significant difference between 4 C and 18 C (p-value>0.05) whereas a significant difference was observed at 25 C where somatic coliphages concentrations start decreasing faster compared to 4– 18 C (p-valueo0.05). In light of this observation, the influence between winter and summer river water sample was studied at 4–18 C (Fig. 3) and at 25 C. Both
A previous publication has shown a difference in behavior between bacteria and viral indicators in the French Moselle river [5]. In this water course, it was observed that thermotolerant coliforms concentrations decreased more rapidly than somatic coliphages concentrations in summer but not in winter. This difference was mainly explained by physical parameters such as temperature and flow rate which depend on the season. According to those results, we decided to investigate batch research at different temperatures in aim to compare the persistence of thermotolerant coliforms, somatic coliphages, and viral genome to the survival of infectious Poliovirus-1 in river water. As infectious Poliovirus-1, used as a model of pathogenic viruses, is not naturally present in the Moselle river, it had to be added to the water samples for this survival study. The relatively high concentration we used (over 4 log ml1) was chosen in order to be able to track Poliovirus-1 for 2 weeks and to mask the possible presence in river water of other cultivable Enterovirus (using BGM cell culture). At the opposite, river water contains naturally thermotolerant coliforms and somatic coliphages so that we did not need to seed the water with these microorganisms. This strategy has the advantage of taking into account the heterogeneous nature of phage and bacteria populations in river water. Indeed, the term ‘‘somatic coliphages’’ groups together several different families of phages (Myoviridae, Siphoviridae, Microviridae, Podoviridae) which do not exhibit the same survival curve in their natural environment. Muniesa et al. [26] demonstrated that Myoviridae are more abundant in an environment recently contaminated by fecal matter (raw or treated wastewater or freshly contaminated river water) while Siphoviridae, which are probably more resistant, predominate in river water far downstream from the pollution. Likewise, the term ‘‘thermotolerant coliforms’’ is a composite term including different bacterial genera (Escherichia, Klebsiella, Enterobacter, Citrobactery). To reach our objective, we chose to work at three different temperatures (4 C, 18 C and 25 C) and with two different river water compositions sampled once in winter and once in summer. Surprisingly, our results show that temperature did not affect significantly the correlations between infectious Poliovirus-1 survival and (potential) indicators persistence with one exception: compared to infectious Poliovirus-1, somatic coliphages concentrations start decreasing faster at 25 than at 4–18 C. Additional data are needed to
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confirm and explain that observation. Concerning the water composition, it appeared different between winter and summer (Table 1). It is interesting to notice that, unlike temperature, this difference affects all correlations between (potential) indicators persistence and infectious Poliovirus-1 survival. Nevertheless, our results underline that the potential contribution of each of these parameters as an indicator of viral contamination is not equivalent and should be examined in detail. According to Armon and Kott [27], the ideal indicator should present a survival curve at least equivalent to that of pathogenic microorganisms. In light of our results, thermotolerant coliforms would appear to be mediocre indicators of the behavior of infectious Poliovirus-1. Thermotolerant coliforms survived less well than infectious Poliovirus-1 in winter river water, irrespective of the temperature used for the survival study. This is in agreement with prior data showing the inefficacy of coliforms as indicators of viral behavior [1–4]. Somatic coliphages were found to survive longer than infectious Poliovirus-1 in all our experimental conditions. They can survive up to five times longer in river water. Such a large difference is particularly interesting since it is known that other enteric viruses (HAV, Norwalk-like virus, rotavirus or adenoviruses 40 and 41) survive longer than Poliovirus-1 [28–33]. Furthermore, these observations throw new light on earlier field work [34,5,35] showing a ‘‘coliphage/coliform’’ ratio less than 1 in river water after recent fecal contamination followed by a rapid inversion of the ratio to more than 1 when measurements are made far from the source of contamination. The longer persistence of somatic coliphages compared with that of thermotolerant coliforms would provide a good explanation of these observations. Skraber et al. [5] also suggested that temperature and flow rate influence the ratio between coliphages and coliforms. This ratio would be greater than 1 when the temperature is high and flow rate low (summer conditions). Our survival curves demonstrate that this inverted ratio can also be observed for low temperature and high flow rate (winter conditions) if the distance from the source of pollution is great enough. Finally, considering the last viral indicator we tested, previous studies have demonstrated that viral genome can persist for a much longer period than the corresponding infectious virus in buffer [11], after chlorination [12] or even in river water [10]. It is therefore very important to evaluate how much more persistence the genome is than the pathogenic virus in river water. Our results show that the viral genome exhibits significantly stronger resistance than the infectious virus. Therefore, it can be assumed that viral genome can be used as a viral indicator in river water based on persistence criteria. The higher persistence of genome compared to infectious Poliovirus-1 can be explained by different mechanisms responsible for the
degradation of the viral capsid (proteasey) and the viral genome (RNasesy). In addition, the degradation of viral RNA can also depend on capsid degradation because of the accessibility of the nucleic acid. Despite of different mechanisms involved in both parameters persistence (genome and infectivity), we observed that the loss of viral genome is directly correlated to the disappearance of infectious virus irrespective of the temperature. So, even if the reason of disappearance is not identical, according to our results, it cannot be excluded that viral genome could be a good viral index (accepting the definition of ‘‘index’’ proposed by [27]) in river water. Nevertheless, as pathogenic enteric viruses have different epidemiological patterns (some occur epidemiologically or endemically or with clear seasonal peak), it clearly appears that the possible monitoring of viral genome in river water should be extend to different genotypes. To achieve this objective, quantitative RTPCR methodology using the 50 non-coding conserved region of viral RNA is already available for the detection of the main Enterovirus [36,37]. It should be also possible to design a similar technique for HAV, Rotavirus, Astrovirus or Adenovirus using some other preserved region. However, for the Norovirus, it would probably be more difficult to develop such a method due to the wide variability of the genome [38]. Quantification of viral genomes would enable monitoring the viral presence in river water environment but viral genome quantification raise the problem of cost which would be much higher than for somatic coliphages or thermotolerant coliforms enumeration. In conclusion, our study confirm that thermotolerant coliforms counts may underestimate viral contamination in river water and shows that quantification of somatic coliphages or viral genome can be used as an indicator of viral survival. Rapid methodology is available for both somatic coliphages and viral genome quantification methods, but special quantitative RT-PCR procedures would have to be designed for multiplex detection of specific pathogenic viruses in the environment. Acknowledgements This work was supported by: *
*
the European Community (TOFPSW: EVK1.2000. 22080); le Conseil Re´gional de Lorraine (ZAbM: Zone Atelier du bassin de la Moselle).
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indicators of the virological quality of water. Water Sci Technol 1986;18:167–73. Lucena F, Araujo R, Jofre J. Usefulness of bacteriophages infecting Bacteroides fragilis as index microorganisms of remote faecal pollution. Water Res 1996;30:2812–6. Tartera C, Jofre J. Bacteriophages active against Bacteroides fragilis in sewage-polluted waters. Appl Environ Microbiol 1987;53:1632–7. Maul A. Aspects statistiques des me´thodes de quantification en virologie. In: Schwartzbrod Lcoordinating editor, editor. Virologie des milieux hydriques. Paris: Lavoisier; 1991. ISO 9308/1. Water quality—detection and enumeration of coliform organisms, thermotolerant coliform organisms and presumptive Escherichia coli, 1990. ISO/FDIS 10705-2. Water quality—detection and enumeration of bacteriophages—Part 2: Enumeration of somatic coliphages, 1999. Muniesa M, Lucena F, Jofre J. Study of the potential relationship between the morphology of infectious somatic coliphages and their persistence in the environment. J Appl Microbiol 1999;87:402–9. Armon R, Kott Y. Bacteriophages as indicators of pollution, Critical reviews. Environ Sci Technol 1996;26:299–335. Biziagos E, Passagot J, Crance JM, Deloince R. Long-term survival of hepatitis A virus and Poliovirus type 1 in mineral water. Appl Environ Microbiol 1988;54:2705–10. Enriquez CE, Hurst CJ, Gerba CP. Survival of the enteric adenoviruses 40 and 41 in tap, sea, and waste water. Water Res 1995;29:2548–53. Gantzer C, Dubois E, Crance JM, Billaudel S, Kopecka H, Schwartzbrod L, Pommepuy M, LeGuyader F. Influence of environmental factors on the survival of enteric viruses in seawater. Oceanol Acta 1998;21:983–92. Keswick BH, Satterwhite TK, Johnson PC, DuPont HL, Secor SL, Bitsura JA, Gary GW, Hoff JC. Inactivation of Norwalk virus in drinking water by chlorine. Appl Environ Microbiol 1985;50:261–4. Qin SM, Gerba CP. Comparative inactivation of enteric adenoviruses, Poliovirus and coliphages by ultraviolet irradiation. Water Res 1996;30:2665–8. Sommer R, Weber G, Cabaj A, Wekerle J, Keckand G, Schauberger G. UV-inactivation of microorganisms in water. Int J Hyg Environ Med 1989;189:214–24. Bell RG. The limitation of the ratio of fecal coliforms to total coliphage as a water pollution index. Water Res 1976;10:745–8. Zaiss U. Dispersal and fate of coliphages in the River Saar. Abt Orig B, Hyg 1981;174:160–73. Donaldson KA, Griffin DW, Paul JH. Detection, quantitation, identification of enteroviruses from surface waters, sponge tissue from the Florida Keys using real-time RTPCR. Water Res 2002;36:2505–14. Monpoeho S, Dehe´e A, Mignotte B, Schwartzbrod L, Marechal V, Nicolas JC, Billaudel S, Fe´rre´ V. Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR. Biotechnology 2000; 29:88–93. Lopman BA, Brown DW, Koopmans M. Human caliciviruses in Europe. J Clin Virol 2002;24:137–60.
Water Research 38 (2004) 2927–2933
Survival of infectious Poliovirus-1 in river water compared to the persistence of somatic coliphages, thermotolerant coliforms and Poliovirus-1 genome S. Skraber, B. Gassilloud, L. Schwartzbrod, C. Gantzer* LCPME–UMR 7564 CNRS-UHP, Faculte´ de Pharmacie, 5 rue Albert Lebrun, B.P. 403, 54001 Nancy, France Received 1 September 2003; received in revised form 8 March 2004; accepted 26 March 2004
Abstract The microbiological quality of water is currently assessed by search for fecal bacteria indicators. There is, however, a body of knowledge demonstrating that bacterial indicators are less resistant to environmental factors than human pathogenic viruses and therefore underestimate the viral risk. As river water is often used as a resource for drinking water production, it is particularly important to obtain a valid estimation of the health hazard, including specific viral risk. This work was conducted to compare the survival of infectious Poliovirus-1 used as a pathogenic virus model to the persistence of, on the one hand, thermotolerant coliforms commonly used as indicators and on the other hand, to somatic coliphages and Poliovirus-1 genome considered as potential indicators. We studied the behavior of infectious Poliovirus-1 and the three (potential) indicators of viral contamination in river water at three different temperatures (4 C,18 C and 25 C). This experiment was performed twice with river water sampled at two different periods, once in winter and once in summer. Our results showed that the survival of thermotolerant coliforms can be 1.5-fold lower than infectious Poliovirus-1. In contrast, under all our experimental conditions, somatic coliphages and Poliovirus-1 genome persisted longer than infectious Poliovirus-1, surviving, respectively, 2–6-fold and about 2-fold longer than infectious Poliovirus-1. According to our results exclusively based on survival capacity, somatic coliphages and viral genome, unlike thermotolerant coliforms appear to be better indicators of viral contamination in river water. Moreover, the disappearance of viral genome is well-correlated to that one of infectious virus irrespective of the conditions tested. r 2004 Elsevier Ltd. All rights reserved. Keywords: River water; Viral indicator; Somatic coliphages; Thermotolerant coliforms; Virus genome; Poliovirus-1; Persistence; Survival
1. Introduction The microbiological quality of rivers used as a drinking water resource is currently evaluated by measuring the density of bacterial indicators (thermo*Corresponding author. LCPME, Faculte´ de Pharmacie, 5 rue Albert Lebrun, B.P. 403, 54001 Nancy, France. Tel.: +3303-83-68-22-91; fax: +33-03-83-68-23-01. E-mail address: [email protected] (C. Gantzer).
tolerant coliforms, Enterococci, spores of sulfite-reducing anaerobes). These fecal indicators should estimate the risk of the presence of enteric pathogens discharged into the river from a wastewater treatment plant upstream from the drinking water source. This objective can be achieved only if the indicator organisms behave in the same way as pathogens in the water flow of the river or during the process of producing drinking water. It has however been largely demonstrated that this prerequisite is not met when bacteria are used as indicators of enteric viruses pathogenic for humans like
0043-1354/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2004.03.041
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Enterovirus, hepatitis virus and viruses that cause gastroenteritis [1–4]. In a recent publication [5], we demonstrated that in the Moselle river, thermotolerant coliforms and Enterococci decrease much more rapidly than fecal viruses represented by somatic coliphages or F-specific phages especially when temperature is high and flow is low (summer period). This observation demonstrates that viral pollution can be underestimated in river water before collection for drinking water production. Different solutions can be proposed to overcome this problem. As suggested by several authors [6,7] direct detection of pathogenic Enterovirus using cell culture might be a possible solution. The fact that this method only detects infectious viruses is an advantage but the delay to results is too long (1–3 weeks) and certain viruses which may be present (Norovirus) would go undetected. The detection of viral genome by reverse transcription polymerase chain reaction (RT-PCR) is a very rapid, sensitive and specific technique. Several authors [8,9] have suggested applying this method, which may detect all viral serotypes, using the viral genome as an indicator of viral contamination. However, the presence of viral genome is not proof of the presence of infectious virus, making interpretation hazardous. In some cases, viral genome can persist much longer than the infectious virus [10–12]. The usefulness of viral genome detection therefore depends on the time interval between loss of infectivity and genome degradation. Fecal bacteriophages are also proposed as indicators of viral contamination. The most frequently studied are somatic coliphages [13–16], F-specific phages [14,17–19] and Bacteroides fragilis phages [14,20–22]. These phages are excreted in human stools and may behave like pathogenic viruses in water. Each of these potential indicators has its own advantages and disadvantages and, since the
ideal universal indicator probably does not exist, should be evaluated individually for each application or protection purpose. Based on our previous observations in natural river water conditions [5], we decided to isolate the temperature parameter and study the survival in river water of infectious Poliovirus-1 used as a pathogenic virus model on one hand, and the survival/ persistence of three (potential) indicators of viral contamination (thermotolerant coliforms, somatic coliphages and Poliovirus-1 genome) on the other hand. Indeed, the aim was, in our experimental conditions, to evaluate the value of those three microbiological parameters as indicators of the presence of infectious Poliovirus-1.
2. Materials and methods 2.1. River water River water samples are taken from the Moselle river in which it has been already observed a difference of behavior between bacterial and viral indicators [5]. In the location called Maron, 40 l were drawn from the middle of the river at a depth of 1 m. Samples were drawn twice: one in September 4, 2001 (summer) and one in January10, 2002 (winter). The microbiological and physicochemical properties of the samples are given in Table 1. 2.2. Poliovirus-1 strain A stock suspension of Poliovirus-1 Sabin (Lsc 2ab) was prepared by inoculating a monolayer of BGM cells. After 24–48 h of virus multiplication, the cells were frozen and thawed three times and centrifuged (10,000 g,
Table 1 Microbiological and physicochemical parameters of the river water samples Parameters
Units
Summer sample
Winter sample
Temperature pH Conductivity Turbidity Hydrogenocarbonates Alkalinity Dry matter at 180 C Aluminum Iron Nitrate Orthophosphate Dissolved oxygen KMnO4 oxidability Suspended solids Thermotolerant coliforms Somatic coliphages
C — mS cm1 NTU Mg HCO3 l1 Mg CaO l1 Mg l1 Mg Al l1 mg Fe l1 Mg NO3 l1 Mg PO4 l1 Mg O2 l1 Mg O2 l1 Mg l1 Log CFU 100 ml1 Log PFU 100 ml1
19.2 8.1 469 6.4 63 29 178 0.150 366 4.4 0.16 9.5 6.12 8 3.10 3.29
1.2 7.7 63 3.7 106 49 202 0.085 95 8.5 o0.05 12.6 1.90 o2 3.84 3.52
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60 min.). The titer of the resulting supernatant liquid which constituted the viral stock was about 8.3 log MPNCU ml1. It was kept at –80 C. 2.3. Quantification of infectious virus by cell culture Each sample was treated with 10% antibiotic and antimycotic solution (Sigma A-5955) for 2 h at 37 C. Quantification of infectious Poliovirus-1 was performed in 96-well microplates using BGM cells. Fifty microliters of three successive logarithmic dilutions for each analyzed sample were added to 200 ml of 7.5 104 cells ml1 in minimum essential medium (MEM: Sigma M-5650) solution containing 2% fetal calf serum. Each dilution was seeded in 40 wells. After 6 days incubation at 37 C under 5% CO2 atmosphere, the plates were examined under microscope and the cytopathogenic effect (CPE) was identified. Wells exhibiting a CPE were counted and the most probable number of cytopathogenic units (MPNCU) was calculated [23]. The concentrations were expressed in MPNCU ml1 and the inactivation in function of time was estimated by the calculation of log N/N0 with N and N0 corresponding to the concentrations of infectious Poliovirus-1, respectively, at time t and 0. 2.4. Viral genome quantification (i) Viral RNA extraction: Poliovirus genome was extracted from 140 ml of sample with the Qiamp viral RNA kit (Qiagen, 52904) according to the manufacturer’s instructions. Hence, 60 ml of final extracted RNA solution were obtained. (ii) Primers and probes: Primers and probes used for Poliovirus-1 detection are presented in Table 2. They were designed using Primer Express 1.0 software and the complete sequence of Poliovirus1 (Genbank, access number: NC 002058). (iii) cDNA synthesis: 5 ml of extracted RNA was mixed with 1 m of 10 mM reverse primer (ENT-r) and heated at 95 C for 3 min. 14 m of a mixture containing 4 m of 5 reverse transcription buffer (250 mM Tris-HCl (pH=8.4), 50 mM MgCl2, 350 mM KCl, 15 mM dithiothreitol, 2.5 mM sper-
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midine) (Promega, M515A), 1 ml of 40 U ml1 RNase inhibitor (Promega, N211A), 2 ml of 10 mM of each deoxynucleoside triphosphate (Perkin Elmer N8080260), 1 ml of 8 U ml1 reverse transcriptase (Promega, M510A), 5 ml Dnase/Rnase free water (Sigma W4502) and 1 ml of 1 mg. ml1 T4gene32 (Roche, 972991) were added. Reverse transcription was performed at 42 C for 60 min. Finally, RNA-DNA hybrids were denatured, and reverse transcriptase was inactivated by heating to 95 C for 5 min. The resulting cDNA was then quantified by PCR. (iv) DNA quantification: PCR assay was performed with 5 ml cDNA and 45 ml of the mixture containing 25 ml Taq man universal Master mix (Perkin Elmer, 4304437), 14.5 ml nuclease free water (Sigma W4502), 0.5 mM forward primer, 0.5 mM reverse primer and 0.5 mM probe. Amplification was performed using an ABI Prism 7700 (Perkin Elmer Inc.). The amplification ramp included two hold programs: (i) 2 min at 50 C to activate the uracil N0 -glycosylase followed by (ii) 10 min at 95 C to release the activity of the Hotstart DNA polymerase. Samples were then submitted to 50 cycles (15 s. at 95 C and 1 min at 60 C). Real-time fluorescence measurements were obtained and directly analyzed by the ABI prism 7700 sequence detection system 1.7.1 software (Perkin Elmer). The threshold cycle (Ct) value generated for each sample was calculated by determining the point at which fluorescence exceeded a threshold limit. The viral genome degradation in function of time was estimated by the calculation of log N/N0 with N and N0 corresponding to concentrations of DNA, respectively, at time t and 0. 2.5. Thermotolerant coliforms Thermotolerant coliforms were quantified on m-FC medium after membrane filtration according to norm ISO 9308/1 [24]. The concentrations were expressed in CFU ml1. The inactivation was estimated by the calculation of log N/N0 corresponding to concentrations of thermotolerant coliforms, respectively, at time t and 0.
Table 2 Primers and probe (Eurogentec) Type Primer Primer Probe a
Name ENT-r ENT-f ENT-p
Reaction RT and PCR PCR PCR
f: FAM reporter dye, t: Tamra quencher dye.
Sequence (50 –30 ) 0
Position 0
5 -CAAGATTGGTTCCTGCTTGATCTT-3 50 -AGCATTGTGATCGATGGCAA-30 50 -f-AGATCTTGGATGCCAAAGCGCTCG-t-30 a
5648-5625 5573-5592 5601-5624
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2.6. Somatic coliphages Somatic coliphages were quantified using the bacterial host strain E. coli (WG5) according to standard methods ISO/FDIS 10705-2 [25]. The concentrations were expressed in PFU ml1. The inactivation was estimated by the calculation of log N/N0 corresponding to concentrations of somatic coliphages, respectively, at time t and 0. 2.7. Experimental protocol Concentrations of thermotolerant coliforms and somatic coliphages naturally present in river water samples are given in Table 1. Since the infectious Poliovirus-1 used as a pathogenic virus model is not naturally found in river water, an artificial contamination was performed. River water sample was divided into three equal aliquots of 5 l. One milliliter of the Poliovirus-1 stock suspension was added to each 5-l river water aliquot. The final concentration was 4.7 log MPNCU ml1 in the summer sample and 4.4 log MPNCU ml1 in the winter sample. The high values of concentration (>4 log) allowed to follow up the survival of Poliovirus-1 for several days under constant stirring at 100 rpm. The three 5-l river water samples were held, respectively, at 4 C, 18 C and 25 C. At the same time, three 5-l river water samples were not contaminated with Poliovirus-1 and were held under the same conditions. These samples were used to monitor survival of the bacterial and phagic indicators in river water as a control to verify if the addition of the Poliovirus-1 suspension that may contain nutriments change the behavior of bacteria or viral indicators. Survival was monitored for 2 weeks. This operation was run once in summer and once in winter. The decline in survival was calculated using the ratio log N/N0 where N0 is the concentration at time 0 and N the concentration at time t: As the determination of concentration for all microbiological parameters has been performed at the same time t; each infectious Poliovirus-1 concentration can be associated to an indicator concentration (pairwised parameters). Thus, the decrease of each indicator could be compared with that of infectious Poliovirus-1 for each temperature (4 C, 18 C and 25 C) and for each sample (winter and summer). Linear regression was used to modelize the log reduction of respectively thermotolerant coliforms, somatic coliphages and Poliovirus-1 genome in function of infectious Poliovirus-1 log reduction. The different slopes were compared using Student’s t test.
3. Results As supposed, we observed that when temperature increases, the persistence of thermotolerant coliforms,
somatic coliphages, infectious Poliovirus-1 or its genome decrease. It was also shown that the addition of the Poliovirus-1 suspension had no significant effect on somatic coliphages survival whereas it changed the behavior of thermotolerant coliforms (data not shown). Therefore, we only considered the survival of thermotolerant coliforms and somatic coliphages in river water samples that had not been spiked with Poliovirus-1. To reach our objective, we compared the persistence of thermotolerant coliforms, somatic coliphages and Poliovirus-1 genome to the survival of the infectious Poliovirus-1 in river water sampled in September (summer) and January (winter) and held at 4 C, 18 C and 25 C. For thermotolerant coliforms, when comparing their survival to the persistence of the infectious Poliovirus-1, temperature did not have a significant effect on the slopes of the linear regressions (p-value>0.05). Thus, summer and winter samples were presented with a single regression equation (Fig. 1). In contrast, pooling the data for the three temperatures demonstrated a significant ‘‘sampling date’’ effect (p-valueo0.05). Thus, survival of culturable thermotolerant coliforms in winter water was lower than that of infectious Poliovirus-1 (slope>1) for the temperature range from 4 C to 25 C whereas the opposite is observed in summer water. When comparing the persistence of the viral genome to that one of the infectious Poliovirus-1, a similar pattern was observed. Indeed, temperature did not have a significant effect on the slopes of the linear regressions (p-value>0.05). Summer and winter samples were thus presented with a single regression equation (Fig. 2). As for thermotolerant coliforms, there was a significant difference in behavior depending on the sampling date (p-valueo0.05) although the difference was relatively
Infectious poliovirus (log N/N0) 0
-1
-2
-3
-4
-5
0 Coliforms (log N/No)
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Summer (4, 18 and 25˚C): y= 0.58x + 0.13 2 R = 0.95
-1
-2
-3
-4
Winter (4, 18 and 25˚C): y = 1.30x + 0.05 R2 = 0.91
y=x
Fig. 1. Relation between the log reduction of infectious Poliovirus-1 and the one of thermotolerant coliforms in two river water (summer } and winter n). Each regression takes into account the results obtained at 4 (white), 18 (gray) and 25 C (black) (ntotal ¼ 49).
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Poliovirus genome (log N/No)
0
-1
-2
-3
-4
-5
0 Winter (4, 18 and 25˚C): y = 0.56 x - 0.02 R2 = 0.97
-1
-2
Summer (4, 18 and 25˚C): y = 0.71 x +0.08 R2 = 0.97 y=x
Fig. 2. Relation between the log reduction of infectious Poliovirus-1 and the one of Poliovirus-1 genome in two river water (summer } and winter n). Each regression takes into account the results obtained at 4 (white), 18 (gray) and 25 C (black) (ntotal ¼ 49). Infectious poliovirus (log N/N0)
-1
-2
-3
-4
-5
Coliphages (log N/No)
0 Summer (4 and 18˚C): y = 0.17x + 0.21 R2 = 0.69
-1 Winter (4 and 18˚C): y = 0.41x + 0.07 R2 = 0.93
-2
comparisons showed a significant sample date effect (pvalueo0.05). The survival of somatic coliphages, in our experimental conditions, was always 2- to over 6-fold higher than that of infectious Poliovirus-1 (slopeo1).
4. Discussion
-3
0
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y=x
-3
Fig. 3. Relation between the log reduction of infectious Poliovirus-1 and the one of somatic coliphages in two river water (summer } and winter n). Each regression takes into account the results obtained at 4 (white) and 18 C (gray) (ntotal ¼ 35). Results obtained at 25 C (black) are indicated (n ¼ 15) but were analyzed separately.
small (slope in summer water=0.71 versus in winter water 0.56). Persistence of Poliovirus-1 genome was always greater than that of infectious Poliovirus-1 (slopeso1). Finally, when comparing the survival of somatic coliphages to that of the infectious Poliovirus-1, analysis of the linear regressions for the three temperatures demonstrated a significant difference in winter water. There was no significant difference between 4 C and 18 C (p-value>0.05) whereas a significant difference was observed at 25 C where somatic coliphages concentrations start decreasing faster compared to 4– 18 C (p-valueo0.05). In light of this observation, the influence between winter and summer river water sample was studied at 4–18 C (Fig. 3) and at 25 C. Both
A previous publication has shown a difference in behavior between bacteria and viral indicators in the French Moselle river [5]. In this water course, it was observed that thermotolerant coliforms concentrations decreased more rapidly than somatic coliphages concentrations in summer but not in winter. This difference was mainly explained by physical parameters such as temperature and flow rate which depend on the season. According to those results, we decided to investigate batch research at different temperatures in aim to compare the persistence of thermotolerant coliforms, somatic coliphages, and viral genome to the survival of infectious Poliovirus-1 in river water. As infectious Poliovirus-1, used as a model of pathogenic viruses, is not naturally present in the Moselle river, it had to be added to the water samples for this survival study. The relatively high concentration we used (over 4 log ml1) was chosen in order to be able to track Poliovirus-1 for 2 weeks and to mask the possible presence in river water of other cultivable Enterovirus (using BGM cell culture). At the opposite, river water contains naturally thermotolerant coliforms and somatic coliphages so that we did not need to seed the water with these microorganisms. This strategy has the advantage of taking into account the heterogeneous nature of phage and bacteria populations in river water. Indeed, the term ‘‘somatic coliphages’’ groups together several different families of phages (Myoviridae, Siphoviridae, Microviridae, Podoviridae) which do not exhibit the same survival curve in their natural environment. Muniesa et al. [26] demonstrated that Myoviridae are more abundant in an environment recently contaminated by fecal matter (raw or treated wastewater or freshly contaminated river water) while Siphoviridae, which are probably more resistant, predominate in river water far downstream from the pollution. Likewise, the term ‘‘thermotolerant coliforms’’ is a composite term including different bacterial genera (Escherichia, Klebsiella, Enterobacter, Citrobactery). To reach our objective, we chose to work at three different temperatures (4 C, 18 C and 25 C) and with two different river water compositions sampled once in winter and once in summer. Surprisingly, our results show that temperature did not affect significantly the correlations between infectious Poliovirus-1 survival and (potential) indicators persistence with one exception: compared to infectious Poliovirus-1, somatic coliphages concentrations start decreasing faster at 25 than at 4–18 C. Additional data are needed to
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confirm and explain that observation. Concerning the water composition, it appeared different between winter and summer (Table 1). It is interesting to notice that, unlike temperature, this difference affects all correlations between (potential) indicators persistence and infectious Poliovirus-1 survival. Nevertheless, our results underline that the potential contribution of each of these parameters as an indicator of viral contamination is not equivalent and should be examined in detail. According to Armon and Kott [27], the ideal indicator should present a survival curve at least equivalent to that of pathogenic microorganisms. In light of our results, thermotolerant coliforms would appear to be mediocre indicators of the behavior of infectious Poliovirus-1. Thermotolerant coliforms survived less well than infectious Poliovirus-1 in winter river water, irrespective of the temperature used for the survival study. This is in agreement with prior data showing the inefficacy of coliforms as indicators of viral behavior [1–4]. Somatic coliphages were found to survive longer than infectious Poliovirus-1 in all our experimental conditions. They can survive up to five times longer in river water. Such a large difference is particularly interesting since it is known that other enteric viruses (HAV, Norwalk-like virus, rotavirus or adenoviruses 40 and 41) survive longer than Poliovirus-1 [28–33]. Furthermore, these observations throw new light on earlier field work [34,5,35] showing a ‘‘coliphage/coliform’’ ratio less than 1 in river water after recent fecal contamination followed by a rapid inversion of the ratio to more than 1 when measurements are made far from the source of contamination. The longer persistence of somatic coliphages compared with that of thermotolerant coliforms would provide a good explanation of these observations. Skraber et al. [5] also suggested that temperature and flow rate influence the ratio between coliphages and coliforms. This ratio would be greater than 1 when the temperature is high and flow rate low (summer conditions). Our survival curves demonstrate that this inverted ratio can also be observed for low temperature and high flow rate (winter conditions) if the distance from the source of pollution is great enough. Finally, considering the last viral indicator we tested, previous studies have demonstrated that viral genome can persist for a much longer period than the corresponding infectious virus in buffer [11], after chlorination [12] or even in river water [10]. It is therefore very important to evaluate how much more persistence the genome is than the pathogenic virus in river water. Our results show that the viral genome exhibits significantly stronger resistance than the infectious virus. Therefore, it can be assumed that viral genome can be used as a viral indicator in river water based on persistence criteria. The higher persistence of genome compared to infectious Poliovirus-1 can be explained by different mechanisms responsible for the
degradation of the viral capsid (proteasey) and the viral genome (RNasesy). In addition, the degradation of viral RNA can also depend on capsid degradation because of the accessibility of the nucleic acid. Despite of different mechanisms involved in both parameters persistence (genome and infectivity), we observed that the loss of viral genome is directly correlated to the disappearance of infectious virus irrespective of the temperature. So, even if the reason of disappearance is not identical, according to our results, it cannot be excluded that viral genome could be a good viral index (accepting the definition of ‘‘index’’ proposed by [27]) in river water. Nevertheless, as pathogenic enteric viruses have different epidemiological patterns (some occur epidemiologically or endemically or with clear seasonal peak), it clearly appears that the possible monitoring of viral genome in river water should be extend to different genotypes. To achieve this objective, quantitative RTPCR methodology using the 50 non-coding conserved region of viral RNA is already available for the detection of the main Enterovirus [36,37]. It should be also possible to design a similar technique for HAV, Rotavirus, Astrovirus or Adenovirus using some other preserved region. However, for the Norovirus, it would probably be more difficult to develop such a method due to the wide variability of the genome [38]. Quantification of viral genomes would enable monitoring the viral presence in river water environment but viral genome quantification raise the problem of cost which would be much higher than for somatic coliphages or thermotolerant coliforms enumeration. In conclusion, our study confirm that thermotolerant coliforms counts may underestimate viral contamination in river water and shows that quantification of somatic coliphages or viral genome can be used as an indicator of viral survival. Rapid methodology is available for both somatic coliphages and viral genome quantification methods, but special quantitative RT-PCR procedures would have to be designed for multiplex detection of specific pathogenic viruses in the environment. Acknowledgements This work was supported by: *
*
the European Community (TOFPSW: EVK1.2000. 22080); le Conseil Re´gional de Lorraine (ZAbM: Zone Atelier du bassin de la Moselle).
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