ABSTRACT
A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45-μm pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H2SO4 (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.
Noroviruses (NVs), formerly called small round-structured viruses or Norwalk-like viruses, have a diameter of 27 to 40 nm and possess a nonenveloped, single-stranded, and positive-sense RNA genome (15). The genus Norovirus is classified in the family Caliciviridae and includes two major groups: NVs of genotype 1 (NV-G1) and NV-G2 (3, 16). NVs are the major agents of acute nonbacterial gastroenteritis in patients of all age groups in both developed and developing countries (15), and they are estimated to be associated with over 90% of acute viral gastroenteritis cases worldwide (10, 14). However, many cases of viral gastroenteritis remain unreported; usually these are sporadic cases, but sometimes large outbreaks also go unreported. This limitation of the surveillance system restricts our understanding of the actual incidence of viral gastroenteritis (4, 8, 17).
NVs are frequently detected in various types of environmental water samples, such as sewage (23, 29), river water (12), well water (5), seawater (20), and even mineral waters (6, 7). NVs have also been detected in shellfish (18, 25-27). There has been one report of detection of NVs in tap water (23), but this was due to a failure in water treatment (i.e., inadequate chlorination). Other enteric viruses, such as poliovirus, coxsackievirus, echovirus, adenovirus, and hepatitis A virus, have been isolated from treated tap water (2, 13, 24, 38), but most of those reports included the procedure of cell culturing, and the detection of noncultivable viruses has been limited. The detection of NVs depends mainly on reverse transcription-PCR because no host cell is available for the cultivation of NVs (43).
In spite of the epidemiological importance of NVs, their behavior in the environment has not been adequately studied. The U.S. Environmental Protection Agency has recommended further study of the family Caliciviridae, including the development of analytical methods for the determination of the environmental occurrence, treatment, and impact on health of its members (41).
In order to understand the fate of enteric viruses in water, sensitive detection methods, especially methods for concentrating a small amount of viruses in water, are required and have been developed by many researchers (30, 37-39, 42, 44). Viruses in water samples are easily adsorbed to a negatively charged filter under the presence of multivalent cations (31). This easy adsorption is likely due to an attractive interaction with the multivalent cations (33). However, this electrical attractive interaction can affect the elution of viruses from the filter; hence, the recovery yield is often very poor. In a previous study, it was shown that rinsing the filter with H2SO4 (pH 3.0) in order to remove magnesium ions and other inhibitory substances improves recovery yields of poliovirus from MilliQ (ultrapure) water and from seawater (20).
Although beef extract (1 to 3%, pH 9 to 11) has often been used to elute viruses, there are organic and inorganic compounds in beef extract known to inhibit cDNA synthesis and PCR amplification (1), which may interfere with the recovery of viruses present in low numbers. To avoid this problem, NaOH (pH 10.8) was used for elution and successful recovery of the viruses (20). However, this method is not suitable for large volumes of freshwater, such as tap water or river water, because it requires the addition of MgCl2 to the sampled water prior to filtration.
In this study, a method for concentrating enteric viruses in large volumes of freshwater was developed and applied to the detection of NVs in tap water in Tokyo, Japan, from January 2002 to February 2003.
MATERIALS AND METHODS
Viruses and cell lines.Poliovirus type 1 (LSc 2ab Sabin strain) was provided courtesy of the Tokyo Metropolitan Research Laboratory of Public Health, Tokyo, Japan, and propagated in the Buffalo green monkey kidney (BGM) cell line in an incubator at 37°C with 5% CO2 (21). BGM cells were grown in Eagle's minimum essential medium (MEM; Nissui Seiyaku, Tokyo, Japan) containing 5% fetal bovine serum (catalog no. 12103-500 M; JRH Bioscience, Lenexa, Kans.), 1% antibiotic-antimycotic (catalog no. 15240-062; Invitrogen, Tokyo, Japan), 1% l-glutamine (29.2 mg/ml; Invitrogen), and 2% sodium bicarbonate solution (7.5%, wt/vol; Invitrogen) in a 75-cm2 tissue culture flask (Iwaki, Tokyo, Japan). The concentration of poliovirus in water was determined by plaque assay using BGM cells propagated in six-well cell culture plates (Corning, Tokyo, Japan). Each sample was diluted with Eagle's MEM containing 1% fetal bovine serum, 1% antibiotic-antimycotic, 1% l-glutamine, and 2% sodium bicarbonate solution, 1 ml of which was added to the cells in each well. After incubation for 90 to 120 min, 3 ml of Eagle's MEM containing 1% fetal bovine serum, 1% antibiotic-antimycotic, 1% l-glutamine, 1.5% sodium bicarbonate solution, and 1.25% agar was overlaid and incubated for 36 to 48 h. The cells were dyed with 0.001% neutral red solution prior to plaque counting.
Water samples.Ultrapure (MilliQ) water and tap water were autoclaved at 121°C for 15 min and inoculated with 1 to 10 μl of poliovirus stock solution (∼107 PFU/ml) to obtain a concentration of 102 to 103 PFU/ml. Sodium thiosulfates were added to the tap water samples for dechlorination prior to the inoculation of poliovirus.
Adsorption of poliovirus to various filters.The efficiency of adsorption of poliovirus to a negatively charged filter with or without multivalent cations was measured and compared to its adsorption to a positively charged filter. Nitrocellulose filters VC, HA, RA, and SM (0.1-, 0.45-, 1.2-, and 5.0-μm pore sizes, respectively, and 47-mm diameter; Millipore, Tokyo, Japan) and GF/F glass filters (0.7-μm pore size and 47-mm diameter; Whatman, Tokyo, Japan) were used as negatively charged filters, and Zetapor glass filters (0.45-μm pore size and 47-mm diameter; Cuno, Meriden, Conn.) were used as positively charged filters. A cation-coated filter was prepared by passing a multivalent cation solution through the filter. Multivalent cation solutions of 2.0 ml of 250 mM AlCl3 and 1.6 ml of 2.5 M MgCl2 were tested. One microliter of poliovirus stock solution (∼107 PFU/ml) was inoculated into 40 ml of MilliQ water, and the sample was filtered through the cation-coated filter. The adsorption yield was calculated based on the concentration of poliovirus present in the filtrate.
Poliovirus recovery from 40 ml of MilliQ water with cation-coated filters with or without an acid rinse.Poliovirus recovery from 40 ml of MilliQ water was measured using a cation-coated filter with or without an acid rinse with 0.5 mM H2SO4 (pH 3.0). Two milliliters of 250 mM AlCl3 was passed through various types of filters with a 47-mm diameter (VC, HA, RA, SM, GF/F, and Zetapor), and 40 ml of MilliQ water inoculated with poliovirus stock solution (∼107 PFU/ml) was filtered. The filter was rinsed with 200 ml of 0.5 mM H2SO4 (pH 3.0) to remove aluminum ions and eluted with 5.0 ml of 1.0 mM NaOH (pH 10.8). The filtrate was recovered in a tube containing 25 μl of 100 mM H2SO4 (pH 1.0) and 50 μl of 100× Tris-EDTA (TE) buffer (pH 8.0) for neutralization.
Poliovirus recovery from 500-ml, 1,000-ml, or 10-liter water samples by a cation-coated filter method.A small volume of poliovirus stock solution (∼107 PFU/ml) was inoculated into 500 ml, 1,000 ml, or 10 liters of MilliQ water or tap water samples and filtered through a 47-mm-diameter HA filter pretreated with 2.0 ml of 250 mM AlCl3. The filter was rinsed with 200 ml of 0.5 mM H2SO4 (pH 3.0) and eluted with 5.0 ml of 1.0 mM NaOH (pH 10.8) into a tube containing 25 μl of 100 mM H2SO4 (pH 1.0) and 50 μl of 100× TE buffer (pH 8.0).
Poliovirus recovery in a reconcentration process with a Centriprep YM-50 filter unit.Centriprep YM-50 ultrafiltration devices (Millipore) were used to reduce the volume of the concentrate. In order to determine the recovery yield, poliovirus stock solution (∼107 PFU/ml) was inoculated into 10 ml of MilliQ water to obtain a concentration of 102 to 103 PFU/ml, followed by ultrafiltration in a Centriprep YM-50 filter unit at 2,500 rpm for 10 min at 4°C to obtain a final volume of approximately 2 ml. The concentration of poliovirus was determined by plaque assay to obtain recovery yields. Four samples were tested.
Determination of the detection limit of poliovirus with the TaqMan PCR system.Poliovirus stock solution (1.4 × 107 PFU/ml) was diluted with MilliQ water (pH 6.3) or beef extract (3%, pH 8.7) by serial 10-fold dilution, and the detection limit for poliovirus was determined using the TaqMan PCR system. RNA was extracted from 140 μl of diluted sample using a QIAamp viral RNA mini kit (QIAGEN, Tokyo, Japan) to obtain a final volume of 60 μl.
Fifteen microliters of extracted RNA was added to a reaction mixture containing 1.5 μl of SuperScript II reverse transcriptase (200 U/μl; Invitrogen), 1.5 μl of 100 mM dithiothreitol (Invitrogen), 6.0 μl of 5× first-strand buffer (Invitrogen), 0.75 μl of 20-U/μl RNase inhibitor (Applied Biosystems, Tokyo, Japan), 1.5 μl of each of the four 2.5 mM deoxynucleoside triphosphate stocks (Applied Biosystems), 1.5 μl of a 50 μM concentration of random hexamers (Applied Biosystems), and MilliQ water to obtain a final volume of 30 μl.
The reaction mixture was incubated at 42°C for 60 min for cDNA synthesis, heated to 99°C for 5 min to inactivate the SuperScript II, and cooled to 4°C with the GeneAmp PCR system 9600 (Applied Biosystems). The resulting cDNA solution was divided into six aliquots of 5 μl to be used as templates for PCR amplification.
Each aliquot was mixed with 45 μl of a reaction buffer containing 25 μl of 2× TaqMan universal PCR master mix (Applied Biosystems), a 400 nM concentration of the sense primer (5′-CCTCCGGCCCCTGAATG-3′), a 400 nM concentration of the antisense primer (5′-ACCGGATGGCCAATCCAA-3′) (36), a 300 nM concentration of the TaqMan probe (5′-FAM [6-carboxyfluorescein]-CCGACTACTTTGGGTGTCCGTGTTTC-TAMRA [6-carboxytetramethylrhodamine]-3′) (20), and MilliQ water to obtain a final volume of 50 μl and was added to a 96-well micro plate (Applied Biosystems). The plate was incubated at 50°C for 2 min and 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min, and it was finally cooled to 4°C.
The ABI PRISM 7200 sequence detection system (Applied Biosystems) was used to determine if the cDNA had been amplified. A normalized reporter value (Rn, defined as the ratio of reporter fluorescent intensity to that of a passive reference) was determined for each tube by using the ABI PRISM 7200 sequence detection system before and after PCR. The magnitude of the signal generated by PCR was calculated as the difference in Rn value (designated dRn). A sample was judged as virus positive when the difference between the dRn value of the no-template control (eight replicates) and that of the sample was significant (P < 0.001).
Collection and concentration of tap water samples for detection of NVs.Ninety-eight tap water samples were collected from the University of Tokyo, Tokyo, Japan, from January 2002 to February 2003 and tested for NVs as shown in Fig. 1. The number of samples taken each month and monthly averages for volume filtered, water temperature, pH, and total chlorine are shown in Table 1. Total chlorine was measured using residual chlorine meters (Sibata Scientific Technology, Tokyo, Japan).
Procedure for detection of NVs in tap water samples.
Numbers of samples and water quality data
One hundred milliliters of 250 mM AlCl3 was passed through an HA filter with a 293-mm diameter using a stainless filter holder (Millipore). The holder was attached with an integrating flowmeter (Aichi Tokei Denki, Aichi, Japan) and connected to the tap directly to allow 100 to 532 liters of tap water to pass through it, followed by filtration of 4,000 ml of 0.5 mM H2SO4 (pH 3.0). Two hundred milliliters of 1.0 mM NaOH (pH 10.8) was used as an elution medium, and the filtrate was recovered in a bottle containing 1 ml of 100 mM H2SO4 (pH 1.0). The resulting virus eluate was reconcentrated as follows. Two milliliters of 250 mM AlCl3 was passed through a 47-mm-diameter HA filter, and 200 ml of the concentrate was filtered again for virus adsorption. The filter was rinsed with 200 ml of 0.5 mM H2SO4 (pH 3.0), followed by viral elution with 5.0 ml of 1.0 mM NaOH (pH 10.8). The filtrate was recovered in a tube containing 25 μl of 100 mM H2SO4 (pH 1.0) and 50 μl of 100× TE buffer (pH 8.0). The concentrates were stored at −20°C until further analysis. After thawing, samples were further concentrated by ultrafiltration (Centriprep YM-50; Millipore) at 2,500 rpm for 10 min at 4°C, for a final volume of approximately 0.9 ml.
TaqMan reverse transcription-PCR for the detection of NVs.RNA was extracted in triplicate from 300 μl of the concentrated sample using SepaGene RV-R (Sanko Jun-yaku, Tokyo, Japan) according to the protocols described by the manufacturer and recovered in a mixture of 12.5 μl of MilliQ water to which 1.0 μl of DNase I (1 U/μl; Wako Jun-yaku, Osaka, Japan) and 1.5 μl of 5× DNase I buffer (1 U/μl; Wako Jun-yaku) was added. The solution was incubated for 30 min at 37°C, followed by inactivation of DNase I for 5 min at 75°C and cooling to 4°C.
All 15 μl of the RNA sample was added to a mixture containing 1.5 μl of SuperScript II (200 U/μl; Invitrogen), 1.5 μl of 100 mM dithiothreitol, 6.0 μl of 5× first strand buffer (Invitrogen), 0.75 μl of a 20-U/μl concentration of the RNase inhibitor (Applied Biosystems), 1.5 μl of each of the four 2.5 mM deoxynucleoside triphosphate stocks (Applied Biosystems), 1.5 μl of a 50 μM concentration of random hexamers (Applied Biosystems), and MilliQ water to obtain a final volume of 30 μl. The reaction mixture was incubated at 42°C for 60 min, followed by 99°C for 5 min and cooling to 4°C.
Each cDNA sample was divided into six samples of 5 μl to obtain 18 aliquots in total from the concentrate; nine aliquots were used for the detection of NV-G1, and nine were used for the detection of NV-G2. Each aliquot was added to a mixture of 25 μl of 2× TaqMan universal PCR master mix (Applied Biosystems), a 400 nM concentration of each primer, a 300 nM concentration of the TaqMan probe, and MilliQ water to obtain a final volume of 50 μl. The reaction mixture was heated at 50°C for 2 min and at 95°C for 10 min. Fifty cycles were then carried out, each consisting of 95°C for 15 s and 56°C for 1 min, followed by cooling to 4°C.
The ABI PRISM 7200 sequence detection system (Applied Biosystems) was used for the detection of NV-G1 and NV-G2. The primer pairs and the TaqMan probes used for the detection of NVs were as follows: for NV-G1, the sense primer was 5′-CGYTGGATGCGNTTYCATGA-3′, the antisense primer was 5′-CTTAGACGCCATCATCATTYAC-3′, and the TaqMan probe was 5′-tetrachloro-6-carboxyfluorescein-AGATYGCGATCYCCTGTCCA-TAMRA-3′;and for NV-G2, the sense primer was 5′-CARGARBCNATGTTYAGRTGGATGAG-3′, the antisense primer was 5′-TCGACGCCATCTTCATTCACA-3′, and the TaqMan probe was 5′-FAM-TGGGAGGGCGATCGCAATCT-TAMRA-3′, where Y is C or T; N is A, C, G, or T; R is A or G; and B is G, T, or C (19).
RESULTS
Adsorption of poliovirus to various filters.Table 2 shows adsorption yields of poliovirus in 40 ml of MilliQ water to six types of filters with or without the addition of multivalent cation solution. In the absence of multivalent cations, adsorption yields decreased with the increasing pore size of the nitrocellulose filters (VC, HA, RA and SM): VC filters showed perfect adsorption of poliovirus, whereas SM filters showed only 37% adsorption. However, the use of an AlCl3 cation coating increased mean adsorption yields from 81, 73, and 37% to 100, 93, and 80% for HA, RA, and SM filters, respectively. It was also effective for GF/F filters, improving adsorption yields from 19 to 85%. In contrast, the adsorption efficiency of Zetapor filters was not improved by the AlCl3 coating (69% for uncoated and 47% for coated filters). Cation coating of HA filters with MgCl2 did not enhance adsorption efficiency.
Poliovirus adsorption efficiency to various filters with or without cations
Poliovirus recovery from 40 ml of MilliQ water via cation-coated filters with or without an acid rinse.Recovery yields of poliovirus from 40 ml of MilliQ water via six types of cation (aluminum ion)-coated filters are shown in Table 3. Over 80% of poliovirus was recovered from HA and RA filters without the acid rinse step. Introduction of the acid rinse step using 200 ml of 0.5 mM H2SO4 (pH 3.0) increased the mean value of poliovirus recovery yields from Millipore nitrocellulose filters (VC, HA, RA, and SM) and GF/F filters. The highest mean recovery yield was obtained by using HA filters, which recovered 99% of poliovirus. The acid rinse step did not improve poliovirus recovery using Zetapor filters, however.
Poliovirus recovery from 40 ml of MilliQ water via a cation-coated filter with or without an acid rinse
Poliovirus recovery from 500-ml, 1,000-ml, or 10-liter water samples by the cation-coated filter method.Table 4 shows the recovery yields of poliovirus from 500 ml, 1,000 ml, and 10 liters of MilliQ water and tap water using cation-coated filters. The mean recovery yields from 500 ml, 1,000 ml, and 10 liters of MilliQ water were 88, 98, and 109%, respectively. High recovery yields were also obtained from tap water samples. Since there was not much difference among the recovery yields from 40 ml, 500 ml, 1,000 ml, and 10 liters of MilliQ water and tap water, increasing the volume to 10 liters per 9.6 cm2 (net area of a 47-mm-diameter filter) did not affect the recovery yields.
Recovery yields of poliovirus from different volumes of water samples
Poliovirus recovery in a reconcentration process using the Centriprep YM-50 filter unit.Seventy-four percent (range, 59 to 91%) of poliovirus inoculated into 10 ml of MilliQ water (2,830 PFU) was recovered by ultrafiltration using the Centriprep YM-50 filter unit. High and stable recovery yields were obtained with an easy treatment compared with those of other reconcentration devices in a short processing time (10 min for four samples).
Detection limit of poliovirus by TaqMan PCR.Table 5 shows the results of detection of poliovirus by TaqMan PCR. In samples diluted with MilliQ water, all five tubes containing RNA of poliovirus equivalent to more than 8.2 × 10−2 PFU were positive and two of five tubes containing 8.2 × 10−3 PFU of poliovirus were also positive, while all five tubes containing 8.2 × 10−4 PFU were negative. Therefore, the detection limit of poliovirus diluted with MilliQ water was between 8.2 × 10−4 and 8.2 × 10−3 PFU per PCR tube. On the other hand, in samples diluted with beef extract, the detection limit of poliovirus per PCR tube was between 8.2 × 10−3 and 8.2 × 10−2 PFU. Thus, the sensitivity of detection can be an order of magnitude lower when beef extract, rather than MilliQ water, is used as an elution medium. Although the precise quantity of poliovirus RNA per tube is not known, the detection limit of 8.2 × 10−4 to 8.2 × 10−3 PFU represents a very high sensitivity obtained by TaqMan PCR.
Detection limit of poliovirus in a PCR tube by TaqMan PCR
Results of detection of NVs in tap water.Over a 14-month period, NV-G1 and NV-G2 were detected in 4 (4.1%) and 7 (7.1%) of 98 samples, respectively. NV-G1 were detected once in July and December 2002 and twice in February 2003, while NV-G2 were detected twice in March 2002, once in April 2002, three times in November 2002, and once in February 2003. NVs were detected more frequently in the winter months (from December to February) than in summer (from June to August): NV-G1 and NV-G2 were detected in 3 (9.4%) and 1 (3.1%) of 32 samples from the winter season, respectively, while only 1 (5.6%) and 0 (0%) of 18 samples from summer was positive for NV-G1 and for NV-G2, respectively. Some tap water samples from the spring season (March to May) were also positive for NV-G2 (3 of 29 samples, 10.3%).
Water quality data for the samples positive for NVs are shown in Table 6. Either NV-G1 or NV-G2 was detected in 9 of 10 samples, and both NV-G1 and NV-G2 were detected in the sample of 1 February 2003. No microbial indicators, such as total coliforms, fecal coliforms, or F-specific RNA coliphages, were measured in this study, but the total amount of chlorine in these samples was over 0.6 mg/liter and met the Japanese standard for drinking water quality. NV-positive samples ranged in volume from 141 to 505 liters; the average was 332 liters, which was similar to the average for all 98 samples. Since each sample was divided into 18 aliquots for PCR, one was equivalent to approximately 10 to 30 liters of tap water. Among the positive results, one of nine aliquots tested was positive for either NV-G1 or NV-G2.
Water quality data for tap water samples positive for NVs
DISCUSSION
In this study, the recovery yields of poliovirus type 1 were determined by plaque assay using BGM cell lines. High adsorption efficiency was obtained when negatively charged filters were precoated with AlCl3 (Table 2), probably because most viruses, including poliovirus, carry negative charges in environmental water at neutral pH (11, 32) and are easy to adsorb to this filter due to the electrical interaction among viruses, aluminum ions, and the filter. The adsorption efficiency was enhanced by the pretreatment with AlCl3 but not with MgCl2, which is probably explained by the valences of the aluminum ion and the magnesium ion; the short contact time of viruses and cations on the filter was not enough for the bivalent magnesium ion to link viruses and the filter. Addition of AlCl3 did not promote the adsorption yields of poliovirus to positively charged Zetapor filters (Table 2), which was consistent with the finding that multivalent cations inhibited the adsorption of viruses to a positively charged filter (31).
Introduction of the acid rinse step using 0.5 mM H2SO4 (pH 3.0) prior to viral elution promoted the recovery of poliovirus from cation-coated glass and from nitrocellulose filters of all pore sizes tested (Table 3). Removal of aluminum ions from the filter was observed, while no poliovirus was eluted from the filter during the acid rinse step (data not shown). Other positively charged ions are likely also removed. It is plausible that the net charge of the viruses turned from negative to positive under acidic conditions such that viruses could remain bound to the filter by direct electrical attractive interaction.
Upon filtration of a small volume of 1.0 mM NaOH (pH 10.8), the charge of viruses turned again to negative so that viruses were released from the filter by repulsive interaction with the negatively charged filter. NaOH elution medium was employed in place of the beef extract to avoid inhibition of enzymatic reactions by organic and inorganic compounds in beef extract (1). We observed a detection limit for poliovirus in beef extract by TaqMan PCR that was an order of magnitude lower than that in MilliQ water (Table 5). It is difficult to compare the detection limits obtained in this study to those reported in other studies, but less than 10 genomes per tube can be detected by TaqMan PCR in general (9, 35).
A Millipore HA filter with a 293-mm diameter was used to filter 100 to 532 liters of tap water. This filter provides 54 times as large a net area (518 cm2) as that of a filter with a 47-mm diameter (9.6 cm2). The method developed in this study could be applied to the concentration of viruses in up to 540 liters of tap water without a decrease in recovery yields, because the recovery yields were high enough from 10 liters of MilliQ water with an HA filter with a diameter of 47 mm (Table 4) and were almost stable with filters with diameters of 47, 90, and 293 mm (data not shown). This method requires no addition of cations or adjustment of pH, and it is suitable for concentrating viruses in a large volume of water. Since the concentration of poliovirus in an experimentally contaminated water sample is much higher than that in natural tap water, it is possible that the cation-coated filter method is less efficient for water containing small quantities of viruses. Moreover, it is not clear if this method sufficiently recovers other enteric viruses, including NVs. Further studies should undertake the evaluation of the recovery of several types of viruses when they are present in low numbers. Although most microorganisms in water can be trapped by filtration on an HA filter with a 0.45-μm pore size, it is possible that some substances in water are concentrated together with viruses or block some pores of the filter, which may inhibit the effective elution of viruses from the filter.
The detection of enteric viruses in tap water has been limited due to the lack of host cells and the low sensitivities of detection methods. In this study, either NV-G1 or NV-G2 was detected in 10 (10.2%) of 98 tap water samples in total (Table 6), and this may be the first study that detected NVs in tap water that had been treated adequately. Although there is no information about the infectivity of NVs detected by PCR, NVs are known to have more resistance to chlorine inactivation than other enteric viruses; they are not inactivated by treatment with free chlorine at a concentration of 0.5 to 1.0 mg/liter (22). Therefore, it might be difficult to inactivate them by the normal chlorination of water supply systems, as the data show that free chlorine levels were 0.6 to 1.1 mg/liter in the NV-positive samples (Table 6).
Some studies imply that the naked viral RNA should be unstable in water and should disappear in a few minutes (28, 40). On the other hand, it is known that viruses inactivated by chlorine can be detected by PCR without any specific treatment (34). We have no data on recovery yields of viral RNA in water using the cation-coated filter method; it is plausible that the naked RNA is not concentrated as efficiently as poliovirus. However, viruses with capsid damage are expected to be concentrated as efficiently as intact viruses, and NVs with no infectivity might have been detected in the present study.
Cell culture PCR, which includes culturing on a host cell prior to PCR, is one of the most effective methods of determining the infectivity of viruses. Active viruses multiply in the cell culturing process and are then easily detected by PCR. However, it is impossible to apply cell culture PCR to the detection of NVs and of some other enteric viruses due to the lack of appropriate host cells. Instead, the TaqMan PCR system was applied to detect NVs in tap water. TaqMan PCR avoids cross-contamination between samples because the caps of reaction tubes need not be opened for electrophoresis after PCR, as the amplification of PCR is observed by in vitro measurement of fluorescence intensity.
By the method developed in this study, NVs were detected in tap water that met the requirement of the Japanese standard for drinking water quality. NVs were detected more frequently in winter than in summer, which agreed with epidemiological reports from the National Institute of Infectious Disease, Tokyo, Japan, which indicated that NVs were more frequently detected in hospitalized patients' feces in the winter. This finding suggests that waterborne infectious diseases caused by NVs in tap water may be a cause for concern.
Although we have examined only NVs in this study, other enteric viruses may also be present in tap water. The World Health Organization, U.S. Environmental Protection Agency, European Communities, and other organizations have referred to enteric viruses in their drinking water quality guidelines, but few of them specify which viruses should be monitored and what levels of viruses are acceptable (13). It is important to quantify the viral-contamination levels in tap water in order to evaluate the risk of viral gastroenteritis and to prevent it.
FOOTNOTES
- Received 13 September 2003.
- Accepted 22 December 2003.
- Copyright © 2004 American Society for Microbiology
