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Wat. Res. Vol. 34, No. 3, pp. 10631067, 2000 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter

TECHNICAL NOTE ULTRAFILTRATION AND REVERSE TRANSCRIPTIONPOLYMERASE CHAIN REACTION: AN EFFICIENT PROCESS FOR POLIOVIRUS, ROTAVIRUS AND HEPATITIS A VIRUS DETECTION IN WATER
HERVE SOULE1*, ODILE GENOULAZ1, BENEDICTE GRATACAP-CAVALLIER1, PERRINE CHEVALLIER2, JING-XING LIU1,3 and JEAN-MARIE SEIGNEURIN1
1

Laboratoire de Virologie Medicale Moleculaire, Faculte de Medecine, CHU, B.P. 217, 38043 Grenoble Cedex 9, France; 2Laboratoire Regional d'Analyses des Eaux, Grenoble, France and 3Department of Microbiology, Second Medical University of Shanghai, Shanghai, People's Republic of China (First received 1 October 1997; accepted in revised form 1 April 1999)

AbstractA process to concentrate viruses from water associated with a rapid and sensitive viral assay was evaluated with water samples experimentally seeded with a single virus or a virus mixture [poliovirus, rotavirus, hepatitis A virus (HAV)]. Tangential ultraltration was used for virus concentration. Reverse transcription-polymerase chain reaction was tested for virus detection and its sensitivity compared to that of cell culture. We recovered the three viruses from experimentally contaminated samples, and detected viral infectivity or RNA with low inputs: 1 TCID50 L1 for cell culture vs 103 TCID50 L1 with the RT-PCR assay in the case of poliovirus, 1 TCID50 L1 in the case of rotavirus whatever the technique, and RT-PCR allowed detection of HAV RNA till at least 1 TCID50 L1. Ninety tapwater samples were also tested for the presence of enterovirus and rotavirus. Five tapwater samples were positive for the RT-PCR assay only: one for poliovirus and four for rotavirus. This procedure allows a control of the virological quality of water. # 2000 Elsevier Science Ltd. All rights reserved Key wordswater, poliovirus, rotavirus, hepatitis A virus, polymerase chain reaction, ultraltration

INTRODUCTION

More than 140 dierent viruses are shed in the gastro-intestinal tract of infected humans, and can be found in waters (Hurst, 1991; Schwartzbrod, 1991). Those viruses generally spread through the fecal oral route, but the importance of viral transmission by water is probably underestimated (Bloch et al., 1990). Poliomyelitis is now becoming very rare, but it was reported by some authors that vaccine-derived viruses could induce paralytic poliomyelitis (Georgescu et al., 1995), or could play a part in some chronic fatigue syndromes (Muir et al., 1993). Rotaviruses are the most important viral pathogens in diarrheal disease of young children. They are believed to spread through water in many outbreaks (Harris et al., 1983; Black et al., 1989). Hepatitis A virus (HAV) is transmitted by contaminated water and food. It causes acute liver disease. Viral concentrations in water are generally low. So, large
*Author to whom all correspondence should be addressed. [Tel.: +33-4-7676-5604; fax: +33-4-7676-5228].

volumes have to be assessed. Many techniques have been described for concentrating viruses contained in water samples, of which are adsorption onto lters (Passagot et al., 1985; Schwartzbrod, 1991; Senouci et al., 1996) or occulation (Kopecka et al., 1993; Ma et al., 1995). With these methods, viruses are trapped and a second step is necessary for their release. Ultraltration has the advantage of concentrating viruses from water samples without any additional steps for their recovery (Divizia et al., 1989; Garin et al., 1993; Gilgen et al., 1997). Two methods can be used to detect viruses in the concentrate. The conventional cell culture was for many years the method of choice for virus detection, but, regard to its sensitivity, the polymerase chain reaction (PCR) technique may be of great interest for environmental virological studies. In this paper, we describe a process to concentrate and detect viruses in water consisting of ultraltration and reverse transcription-polymerase chain reaction (RT-PCR), we compare RT-PCR to the conventional cell culture, and then, we apply the whole described process to tapwater samples.

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SAMPLES AND METHODS

Technical Note the CRSSA, Grenoble, France. Both concentrates of samples contaminated with a mixture of viruses and of tapwater samples were just tested by the RT-PCR assay. Nucleic acid detection using RT-PCR RNA extraction. Extraction (Chomczynski and Sacchi, 1987) was carried out after the second concentration. A denaturing solution containing guanidinium thiocyanate (0.3 mL), 0.8 mL of phenol (water saturated) and 0.2 mL of chloroformisoamyl alcohol mixture (49:1) were added sequentially to the concentrate, with thorough mixing after the addition of each reagent. The nal suspension was vortexed for 10 s and cooled on ice for 15 min. Samples were centrifuged at 10,000g for 15 min at 48C. The upper aqueous phase was transferred to a sterile Eppendorf tube and 25 ml of silica (Sigma) prepared according to Boom et al. (1990) were added in the tube which was then agitated on a rotating mixer for 20 min. The sample was then centrifuged for 15 s, and the supernatant was discarded. The pellet was washed two times with a buer containing 50% ethanol; 10 mM TrisHCl (pH 7.4); 1 mM EDTA; 50 mM NaCl. A nal washing was performed with 70% ethanol. The pellet was suspended in 50 mL of diethylpyrocarbonate (DEPC) treated water and heated for 10 min at 568C to elute RNA from the silica. After centrifugation, the supernatant was collected and kept at 808C until batch processing. Primers for RT-PCR. For PV (and other enteroviruses), primer sequences were selected from highly conserved 5' non-coding region having a high degree of homology with the known enteroviral RNA sequence (Rotbart, 1990): oligonucleotides sens 5'-CCTCCggCCCCTgAATgCggCTAAT-3' and anti-sense 5'ATTgTCACCATAAgCAgCCA-3' bracketing a 154 bp long fragment. In the case of RV, specic primers in gene segment 9 which encodes the VP7 protein were selected to produce a full length copy of this gene (Gouvea et al., 1990): oligonucleotides sens 5'-ggCTTTAAAAgAgAgAATTTCCgTCTgg-3' and anti-sense 5'-ggTCACATCATACAATTCTAATCTAAg-3' bracketing a 1062 bp long fragment. These primers were selected because their sequences are highly conserved among all group A rotaviruses. And, for HAV, primers were situated within the conserved region VP3VP1 of the genome (Margolis and Nainan, 1990): oligonucleotides sens 5'gTTTTgCTCCTCTTTATCATgCTATg-3' and anti-sense 5'-ggAAATgTCTCAggTACTTTCTTTg-3' bracketing a 248 bp long fragment. Reverse transcription (Table 1). Reverse transcription (RTr) was carried out with 10 mL of resuspended RNA, denaturated in a heating block according to table 1 protocols and immediatly cooled on ice. A RTr mixture (10 mL) containing deoxynucleoside triphosphates (10 mM each), 10 pmol anti-sense primer, 20 U of ribonuclease inhibitor (Boehringer), 4 mL of 5 reverse transcriptaseGibco BRL buer, 1 mL of solution Hepes/0.1 M hydrochloride pH 6.9, 2 mL 100 mM dithiothreitol and 200 U of Moloney murine leukemia virus reverse transcriptase (MMLVRTGibcoBRL) was added to each denatured RNA sample tube. In the case of water samples contaminated with a virus mixture, anti-sense primers for PV, RV and HAV were used independently in three dierent tubes, and for tapwater samples, HAV RT-PCR was not performed. The samples were then subjected to one cycle of RTr. Polymerase chain reaction (Table 1). PCR was carried out in a nal volume of 50 mL. Ten mL of each cDNA product were mixed with 40 mL of the PCR reaction mixture containing 10 mM TrisHCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl2; 0.01% gelatin; 200 mM of each dNTP; 40 pmol of sens and anti-sense primers and 1.25 units of Taq DNA polymerase (Perkin Elmer Cetus). Samples

Water samples Samples of sterile distilled water (2 L) were seeded with 2 mL of a known concentration of poliovirus Sabin type 1 (PV) (10-fold dilutions of 106 TCID50 mL1) or rotavirus bovine strain RFC 67 (RV) (10-fold dilutions of 105 TCID50 mL1). Three samples were also simultaneously contaminated with PV, RV and the hepatitis A virus strain CF53 (HAV). The titres of PV, RV and HAV were 10, 103 and 102 TCID50 L1, respectively in the rst mixture, 1, 102 and 10 TCID50 L1 in the second and 1 TCID50 L1 for the three viruses in the third. Ninety tapwater samples (2 L) were also studied. They were collected in the region Isere (France), 10 during spring and 80 during winter. The rst 10 were chosen for their known frequent contamination with indicator bacteria. The other samples were collected during an epidemiological survey of infantile rotaviral gastroenteritis (Soule et al., 1999): 56 at the home of children with rotaviral diarrhea and 24 in three towns, at the same place, once a week during 8 weeks. Tapwater samples were also tested for the presence of indicator bacteria according to the guidelines from the Association Francaise de Normalisation. Negative control samples (sterile distilled water) were included in all experiments. Sample concentration Concentration was performed with the Minitan1 system (Millipore), which uses four polysulfonate membranes (cat. PTHK, 105 nominal molecular weight limit, Millipore) for tangential ultraltration. The samples were kept in ice for 20 min during the ultraltration period. The procedure was stopped when no more water was present in the sample reservoir, and the concentrate was the dead volume of the Minitan1 system, approximately 15 mL. After each ltration, the membranes were alternatively treated with 0.1 M HNO3 and 0.1 M NaOH, and thoroughly rinsed with sterile water before each run. Each concentrate was separated into two parts; the rst was kept for cell culture analysis and the second was concentrated again before RT-PCR analysis. In the case of samples seeded with a single virus, ultracentrifugation was chosen for the second concentration step: after a 4 h ultracentrifugation at 75,000g, 48C, the pellet was recovered in 0.3 mL guanidinium buer. For water samples contaminated with a virus mixture and tapwater samples, the Centriprep1 concentrator (Amicon) was used for the second concentration, and the samples concentrated to about 0.5 mL. Centriprep1 employs Amicon's YM membrane for ultraltration, with a cut-o of 105 Da. The concentrators had been sterilised, and just before ultraltration, the membranes were treated with a 3% beef extract solution. Cell cultures The detection of viruses in the concentrates was performed by using cell culture assays. Their sensitivity has been determined in the case of samples contaminated either with PV or with RV. MRC5 and MA104 cells were grown on 6-well plastic plates (Falcon). In the case of PV detection, MRC5 cells were inoculated with the concentrate, whereas for RV, the culture was performed with MA104 cells after addition of trypsin to the concentrate (trypsin Sigma IX 0.5 mg per mL). Cells were incubated at 378C, in an atmosphere enriched with 5% CO2, and observed daily. The culture assay was considered positive when a cytopathic eect appeared under microscopic examination, and was conrmed in a subculture. No cell culture was performed for HAV. HAV suspension had been previously titrated by cell culture on PLC/PRF/5 cells (human hepatocarcinoma) and kindly provided by

Technical Note
stop reaction +48C +48C +48C

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were overlaid with mineral oil and incubated in a thermal DNA cycler (Perkin Elmer Cetus) (Table 1). Agarose gel analysis: hybridization. The nal RT-PCR products were visualised by ethidium bromide staining and exposure to ultraviolet light after gel electrophoresis in 2% agarose. Then, RT-PCR products were transfered to a nylon membrane. After 1 h incubation in prehybridization buer at 408C under agitation, a specic internal 32 P labelled probe was added (5'-ATgAAACCCACAggCACAAAg-3', nucleotides 548 to 568, for PV and other enteroviruses, 5'-gATCCTgTTggCCATCC-3', nucleotides 376 to 392, for RV and 5'-TCAACAACAgTTTCTACAgA-3', nucleotides 2232 to 2251, for HAV). Hybridization lasted 2 h at 408C, and was followed by three washings of 10 min each, successively with SSC 2 , SSC 2 SDS 1% and SSC 0.2 SDS 0.1%. X-ray lms were exposed to the dried lters overnight at 808C.

nal extension extension annealing

558C, 1 min 508C, 2 min 558C, 1 min

728C, 1 min 728C, 2 min 728C, 1 min

728C, 8 min 728C, 8 min 728C, 8 min

RESULTS

Sensitivity was evaluated after the addition of known concentrations of PV (2 106 to or RV (2 105 to 2 103 TCID50) 2 2 10 TCID50) into 2 L samples of distilled sterile water. According to our experiments, after concentration with the Minitan1 system, the smallest detectable inoculum in the culture assay was 1 TCID50 L1 both for PV and RV. After two steps of concentration, the RT-PCR analysis allowed the detection of 103 TCID50 L1 PV RNA (Fig. 1). As regard RV, the sensitivity of RT-PCR was similar to but not higher than cell culture (1 TCID50 L1) (Fig. 1). In the assays with virus mixtures, after two steps of ultraltration, the three viruses were simultaneously recovered from experimentally contaminated samples, and RT-PCR allowed selective detection of PV, RV and HAV (Fig. 1) up to 1 TCID50 L1 for each virus. We have carried out some assays with samples containing PV, RV or HAV and humic acid (Sigma). Samples with at least 100 mg/mL humic acid lead to false negative PV, RV and HAV RTPCR results, even with high virus amounts. If RNA extraction included a purication step with silica particles, the inhibitory eect of humic acid on RT-PCR was observed for a concentration ten times higher (1 mg/mL). Five of the 90 tapwater samples (5.6%) were virus-positive, whereas nine contained indicator bacteria (10%). Viruses (enteroviruses and RV) and indicator bacteria were never detected simultaneously in a same sample. Among the 10 samples collected during spring, one was positive for enterovirus RT-PCR; all the 10 were RV RT-PCR negative. Conversely, among the 80 samples collected during winter, four were positive for RV RT-PCR and all the 80 were enterovirus RT-PCR negative. The rotavirus-positive samples had been collected at the home of children with rotaviral gastroenteritis. Abundant rainfalls had occurred before collection of four of the ve samples detected virus positive.

Table 1. RT-PCR protocols for enterovirus, rotavirus and hepatitis A virus

denaturation cycle number RNA denaturation cDNA transcription RT Virus type PCR

Enterovirus Rotavirus Hepatitis A virus

958C, 3 min 978C, 5 min 988C, 3 min

378C, 90 min (inactivation: 958C 4 min, stop reaction at +48C) 378C, 90 min (inactivation: 958C 4 min, stop reaction at +48C) 378C, 90 min (inactivation: 958C 4 min, stop reaction at +48C)

35 35 40

948C, 1 min 948C, 1 min 948C, 1 min

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Technical Note

Fig. 1. Detection of (1) poliovirus 1 (10103 TCID50 L1), (2) rotavirus (101 TCID50 L1) in experimentally contaminated water samples and of (3) hepatitis A virus (10 or 1 TCID50 L1) in water samples contaminated with a virus mixture. Two-liter samples of experimentally contaminated water (one virus poliovirus, rotavirus- or a virus mixture HAV+poliovirus+rotavirus-) were treated as described in Materials and Methods and subjected to RT-PCR. Stained lter after Southern blot hybridization with a specic internal 32P labelled probe.

Three of them had been submitted to conventional drinking water treatment process. The specicity of PV, RV and HAV RT-PCR has been controlled by testing RV and HAV primers with PV RNA, PV and HAV primers with RV RNA, or PV and RV primers with HAV RNA; RT-PCR were regularly negative, whatever the amount of viral RNA tested. PV, RV and HAV RT-PCR of control samples gave negative results in all cases.

DISCUSSION

We have tried to develop a process to detect simultaneously by RT-PCR several viruses frequently present in environmental water samples. Large water samples are generally required for virological analysis, because of the low virus inputs (Ma et al., 1995; Senouci et al., 1996). In our study, we used 2L samples, because it is approximately the average daily consumption of water by humans. Ultraltration can be used as a rst or a second step in the concentration process (Divizia et al., 1989; Garin et al., 1993; Shieh et al., 1995). This method needs neither acidication nor addition of polycation salts to water. Moreover, this technique, which does not depend on virus adsorption to a matrix, minimises the loss of virus. In the case of poliovirus, a pretreatment of the lters with beef extract has been found to increase the number of positive results (Divizia et al., 1989). In our study, by pretreating only the secondary ultraltration system, we have recovered simultaneously all three viruses, even at low inputs. In the case of virus mixtures, we have not tested titres lower than 1 TCID50 L1, since the aim of our experiments was to reproduce conditions in which persons might be contaminated. Quite similar results are reported by

other authors for coxsackievirus and HAV after ultraltration and RT-PCR (Gilgen et al., 1997). Detection of poliovirus, rotavirus or HAV in the concentrates was performed by using a RT-PCR assay, and cell culture method has also been used for detection of poliovirus or rotavirus. Inoculation of cell culture and viral replication within cells are the best way of demonstrating an infectivity in the viral concentrate. A cytopathic eect can be assessed by microscopic examination (Hurst et al., 1989). However, cell cultures have three disadvantages: certain viruses cannot grow in vitro, each virus or group of viruses often needs a specic type of cell culture, nally a long time is sometimes required until results are available. Conversely, although RT-PCR cannot reveal the infectivity of viral samples, it can be used for detection of noncultivable viruses, and very low viral inputs can be detected. Moreover, semiquantitative results can be obtained (Hurst et al., 1989). RT-PCR can thus be considered as a tool of great interest for revealing the virological safety of environmental waters, though very small samples only can be analysed. So, a multi-steps concentration is often needed to reduce environmental water samples, and the main disadvantage of RT-PCR is the sensitivity of the transcriptase and polymerase to the presence of inhibitors which can also be concentrated with viruses (Santos and Gouvea, 1994; Ijzerman et al., 1997). Therefore, during the extraction step, it is very important to purify nucleic acids with products like cellulose bers or silica particles (Boom et al., 1990; Puig et al., 1994). As regard the results of our experiments with humic acid, and since concentrations in surface waters have been estimated to 300 mg/mL (Croll, 1972), silica particles insure eciency of RNA purication and RT-PCR. Besides, we have applied the process described hereby (ultraltration, RNA purication with silica

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particles and RT-PCR) to tapwater samples, and detected rotavirus or enterovirus in 5.6% of them. Contamination with indicator bacteria and viruses were never correlated, as previously reported by others (Keswick et al., 1984). Forty-nine samples collected were treated waters. Viruses were detected as often in treated (3/49) and non-treated (2/41) waters. Conversely, indicator bacteria were found in 17% of non-treated waters (7/41) and 4% of treated waters (2/49). This result suggests that commonly used treatments for potable water are less ecient to remove viruses, especially rotaviruses, than bacteria. Current microbial standards used as safety criteria for water may not always be indicative of viruses. Therefore an additional indicator of the virological quality of water is required. The process described hereby to concentrate viruses from environmental waters may be applied to the great majority of viruses, because it does not need chemical reactives for their recovery, which can be toxic for some of them. So this method may be a useful tool in the control of water quality in order to protect the health of populations.
AcknowledgementsWe thank M. Aymard (Lyon) who kindly provided us with poliovirus type 1 stocks (SABIN strain), J. Cohen (INRA, Paris), for the rotavirus bovine strain RFC 67, and R. Deloince (CRSSA, Grenoble) for the hepatitis A virus strain CF53. We also thank F. Leveque (CRSSA, Grenoble) for helpful discussions on HAV PCR.
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