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. 2009 Jan 5;75(5):1264–1270. doi: 10.1128/AEM.01166-08

Continuous Presence of Noroviruses and Sapoviruses in Raw Sewage Reflects Infections among Inhabitants of Toyama, Japan (2006 to 2008) ▿

Masae Iwai 1,*, Sumiyo Hasegawa 1, Mayumi Obara 1, Kazuya Nakamura 1, Eiji Horimoto 1, Takenori Takizawa 1, Takeshi Kurata 1, Shun-ichi Sogen 2, Kimiyasu Shiraki 3
PMCID: PMC2648165  PMID: 19124591

Abstract

Various genotypes of norovirus (NoV) (genogroup I genotype 1 [GI.1], -2, -4, -5, -8, -11, -12, and -14; GII.3, -4, -6, -7, -10, -13, -14, and -15), and sapovirus (SaV) (GI.1 and GI.2, GII.1, and GIV.1) were detected from raw sewage from April 2006 to March 2008, while limited numbers of genotypes of NoV (GI.8, GII.4, GII.6, and GII.13) and SaV (GII.3 and GIV.1) and of NoV (GII.4, GII.7, and GII.13) were detected from clinical cases and healthy children, respectively. During the winter 2006 to 2008, a large number of sporadic gastroenteritis outbreaks and many outbreaks caused by NoV GII.4 occurred among inhabitants in Toyama, Japan. The copy number of genomes of NoV GII detected from raw sewage changed in relation to the number of outbreaks. NoV strains of the same genotypes observed in both raw sewage and human specimens belonged to the same cluster by phylogenetic analysis and had almost identical nucleotide sequences among each genotype. These data suggest that NoVs and SaVs detected from raw sewage reflect the viruses circulating in the community, irrespective of symptoms, and that subclinical infections of NoV are common in Japan. Combined surveys of raw sewage with those of clinical cases help us to understand the relationship between infection of these viruses and gastroenteritis.

Norovirus (NoV) and sapovirus (SaV), members of the Caliciviridae family, are considered to be a major cause of acute gastroenteritis in humans. Both NoV and SaV infect humans via the fecal-oral route and cause family or community-wide outbreaks, mainly in the winter season. NoVs are shed in feces at a level of 105 to 109 virus particles per gram during the symptomatic phase (32, 37), and viruses are continuously shed from patients after cessation of the symptoms (28, 37, 40). In addition, recent reports showed relatively high levels of shedding of the viruses from asymptomatic individuals (7, 8, 32, 37).

NoVs and SaVs show high diversity in their genomes (5, 9). According to such a genetic diversity, they are classified into several genogroups (genogroup I [GI], GII, and GIV for human NoV and GI, GII, GIV, GV for human SaV) and further divided into many genotypes (NoV GI genotypes 1 to 14 [GI.1-14] and GII.1-17 and SaV GI.1-5, GII.1-6, GIV.1, and GV.1) (10, 17, 18). In 2006 to 2007, NoV GII.4 caused a large number of outbreaks of acute gastroenteritis worldwide (1, 11, 35, 43, 45). However, the other genotypes of NoV and SaV may infect humans asymptomatically and persist in the environment.

Raw sewage could contain enteric viruses shed from affected people, and therefore, detectable viruses in raw sewage would reflect the actual state of the circulating viruses in the area. We previously reported that polioviruses in raw sewage and river water were isolated at the same time as oral vaccination in babies, and these isolates were derived from vaccine strains (13, 30). We also showed that the nucleotide sequences of echovirus type 13 isolated from river water were closely related to those from patients with aseptic meningitis during the outbreak in 2002 (14). For NoVs and SaVs, many epidemiological surveys have been conducted to determine the prevalence and virological properties of these viruses (42). Previous reports have shown that the nucleotide sequences of NoV strains from stools of outbreaks in nursing homes and from sewage were identical for an individual outbreak (26), and NoVs detected from gastroenteritis patients, domestic sewage, river water, and cultivated oysters in the area were related to each other (44). However, less is known about infection of the viruses with minor genotypes that are silently circulating in the population.

In this study, we investigated NoVs and SaVs in raw sewage from 2006 to 2008 in Japan and compared the results with the viruses detected from clinical cases as well as healthy individuals to show the comprehensive prevalence of these viruses in the community.

MATERIALS AND METHODS

Samples and preparation of viral suspension. (i) Raw sewage.

Raw sewage was collected monthly from April 2006 to March 2008 at the threshold point of a waste tank in the sewage disposal plant located in Toyama Prefecture, Japan. This facility covers an area with about 300,000 inhabitants, which is the largest group served by 29 sewage disposal plants for a population of 1,100,000 in Toyama Prefecture. The raw sewage from each household in the area reaches the facility within 4 to 8 h. The temperature of raw sewage ranged from 13.8°C to 25.0°C during the year. The average inflows of raw sewage per day in the fiscal year 2007 were 46,063 m3 (70.9%), 2,535 m3 (3.9%), and 16,323 m3 (25.2%) derived from household sewage, industrial wastewater, and unidentified wastewater, respectively.

Two liters of raw sewage was centrifuged at 3,000 rpm for 30 min (4°C), and the supernatants were applied for subsequent concentration of viruses using the filter adsorption and elution method and the polyethylene glycol (PEG) precipitation method, as described previously (24, 29).

For the filter adsorption and elution method, MgCl2 (final concentration, 0.05 M) was added to 1 liter of the supernatant of the raw sewage, and the pH was adjusted to 3.5 with HCl. The supernatant was filtered through a mixed cellulose ester-type membrane to adsorb viruses. Then, the membranes were soaked in 10 ml of 3% beef extract solution, and the viruses were eluted by sonication.

For the PEG precipitation method, 80 g of PEG 6000 (WAKO) was added to 1 liter of the supernatant of raw sewage, and the supernatant was stirred for 2 h at 4°C to suspend PEG 6000. After the suspension was centrifuged at 10,000 rpm for 30 min (4°C), the pellet was collected and dissolved in 4 ml of 0.15 M Na2HPO4 (pH 9.0). The solution was recentrifuged at 10,000 rpm for 30 min (4°C) to recover the supernatant. The eluted solution or the recovered supernatant was used for viral RNA extraction.

(ii) Stool specimens.

A total of 805 fecal specimens were collected from hospital patients with gastroenteritis or from outbreaks from April 2006 to March 2008. An outbreak was defined as occurring when at least three patients from the same area came down with similar clinical symptoms at roughly the same time. A total of 780 of the 805 samples could be assigned to 59 outbreaks according to the above definition. The remaining 27 samples were considered cases of sporadic gastroenteritis diagnosed at pediatric clinics.

With permission from guardians, 134 fecal specimens were collected from healthy children from 2006 to 2008. The ages of the healthy children ranged from <1 to 6 years old. The specimens were originally collected for a survey of poliovirus in September and January in 2006 to 2008, which is at least 2 months after the vaccination for poliovirus. Personal information of donors was disconnected and deidentified from the samples.

A 10% (wt/vol) suspension of stool was prepared by mixing with phosphate-buffered saline, followed by centrifugation at 13,000 rpm for 30 min (4°C). The supernatant fluids were used for viral RNA extraction.

Reverse transcription-PCR and genotyping.

Viral RNA was extracted from 140 μl of concentrated raw sewage or supernatant of stool suspension, using a QIAamp viral minikit (Qiagen) according to the manufacturer's procedure. Extracted RNA was treated with 5 U of DNase I (TaKaRa), and cDNA was synthesized by SuperScript III reverse transcriptase (Invitrogen) with a random hexamer, according to the manufacturer's instructions. The cDNA was used for PCR and real-time PCR.

For NoV PCR, we used the primers SK1F (sense; 5′-CTG CCC GAA TTY GTA AAT GA-3′), SK1R (antisense; 5′-CCA ACC CAR CCA TTR TAC A-3′), SK2F (sense; 5′-CNT GGG AGG GCG ATC GCA A-3′), and SK2R (antisense; 5′-CCR CCN GCA TRH CCR TTR TAC AT-3′), which amplify 330 bp corresponding to nucleotides (nt) 5342 to 5671 of Norwalk/68/US (GenBank accession number M87661) for GI or 344 bp (nt 5046 to 5389) of Lordsdale/93/UK (accession no. X86557) for GII, encompassing the 3′ end of ORF1 to the beginning of the capsid region, as described by Kojima et al. (21). For SaV PCR, we used the primers SV-F11 (sense; 5′-GCY TGG TTY ATA GGT GGT AC-3′) and SV-R1 (antisense; 5′-CWG GTG AMA CCA TTK TCC AT-3′), which amplify about 780 bp (nt 5098 to 5878 of Manchester virus; accession number X86560) of the N-terminal of VP1 region, as described by Okada et al. (36). To determine the genotypes of these viruses, the PCR products were directly applied for sequence analysis using an ABI Prism BigDye Terminator, version 3.1, cycle sequencing kit and an ABI Prism 3100 DNA sequencer (Applied Biosystems). TA cloning (TOPO TA cloning kit; Invitrogen) of the NoV GII PCR products derived from raw sewage from July to October in 2006 was also performed to detect mixed populations of NoV GII.4 because the predominant genotype of NoV GII.4 had changed from 2006a and Chiba-4e to 2006b during that time, as described in the Results section. We sequenced 25 amplicons per month. The genotypes were determined by comparing sequences with those of reference strains in the GenBank (6, 10, 17, 18, 25, 35, 41, 42). The genetic relationship between the strains in this study (Toyama strains) and reference strains was analyzed by MEGA, version 3.1, software (23), using the genomic regions described above. The phylogenetic tree was constructed by the neighbor-joining method after estimation of genetic distance using the Kimura two-parameter method. A bootstrapping test was performed 1,000 times.

Real-time PCR.

The cDNA samples of NoV were used for the TaqMan-based real-time PCR as previously described by Kageyama et al. (16). NoV probes labeled with the TaqMan dye VIC were synthesized at Applied Biosystems Japan, Ltd.

Counting of coliforms.

The amounts of coliforms in raw sewage were measured using the pour plate method with desoxycholate agar (39). Briefly, each 1 ml of a 10-fold dilution of raw sewage was spread into the 90-mm-diameter dishes and then mixed with 20 ml of desoxycholate agar medium. The dishes were incubated at 35°C overnight. The number of colonies of coliforms was counted and defined as the number of CFU.

Statistical number of pediatric patients with gastroenteritis.

The number of patients with gastroenteritis was counted according to the reports from 29 out of 141 pediatric clinics (20.6%) in Toyama Prefecture, from April 2006 to March 2008.

Nucleotide sequence accession numbers.

Nucleotide sequences determined in this study were deposited in the GenBank database under accession numbers AB437141 to AB437235.

RESULTS

NoVs and SaVs in raw sewage.

NoVs and SaVs detected from raw sewage from April 2006 to March 2008 are summarized in Fig. 1. Both the GI and GII genogroups of NoVs were observed almost every month during the survey period. They included eight genotypes of GI (GI.1, -2, -4, -5, -8, -11, -12, and -14) and eight genotypes of GII (GII.3, -4, -6, -7, -10, -13, -14, and -15). NoV GII.4 was the most frequently detected among the genotypes of NoVs, especially in winter and spring. NoV GI.4 was detected with the second highest frequency. Four genotypes of SaVs, GI.1, GI.2, GII.1, and GIV.1, were observed, and GI.1 was the most frequently detected among four genotypes. The frequencies of detection of NoVs GI.4, GII.4, and SaV GI.1 were significantly high between each genotype of NoV GI, NoV GII, and SaV, respectively (χ2 test, P < 0.001).

FIG. 1.

FIG. 1.

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Genotypes of NoV and SaV detected from raw sewage (2006 to 2008). Shading indicates times of virus detection. Frequency of virus detection (*) is defined as the ratio of the number of months when viruses were detected to total number of months in the investigation period.

Thus, various genotypes of NoV and SaV were detected from raw sewage, while NoV GII.4 was predominantly detected in winter and spring.

Quantification of NoVs in raw sewage.

We quantified the NoVs in raw sewage using real time PCR to examine the seasonal changes. To concentrate viruses, both the filter adsorption and elution method and the PEG precipitation method were employed since efficiency or preference of virus detection is believed to be different between these two methods. Although the filter adsorption-elution method was found to be more sensitive than the PEG precipitation method, both methods showed similar profiles of seasonal change (Fig. 2).

FIG. 2.

FIG. 2.

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Quantification of NoV in raw sewage. (A) Values indicate the number of NoV GI genome copies per liter of raw sewage concentrated by the filter absorption and elution method (filter) and the PEG precipitation method. (B) Values are the number of NoV GII genome copies per liter of raw sewage concentrated by each of the indicated methods. The dotted lines indicate the amounts of coliforms per milliliter of raw sewage.

The raw sewage contained not only human excrement but also drainage, such as from a factory; therefore, the degree of dilution of raw sewage might differ every day. Therefore, we measured the number of coliforms as an index for the dilution level of raw sewage. The number of coliforms in raw sewage ranged from 2.0 × 104 to 8.1 × 105 CFU/ml, and the geometric mean titer was 2.0 × 105 CFU/ml (Fig. 2). The number of coliforms showed only a small dispersion (coefficient of variation [CV], 0.076) during the survey period. It indicates that the input of raw sewage into the sewage disposal plant is almost uniform. Furthermore, there was no correlation between levels of coliforms and either NoV GI or NoV GII (r = −0.0751 or r = −0.0331, respectively; P < 0.05).

The copy number of NoV GI in raw sewage ranged from 4.6 × 102 to 2.3 × 106 copies/liter (CV, 0.17) during the survey period, except for October 2006, when NoV GI was not detected. Although smaller amounts of NoV GI in raw sewage were observed than NoV GII, as described later, sewage tended to contain larger amounts of NoV GI in the period from winter to spring than from summer to fall (Fig. 2A).

The copy number of NoV GII ranged from 3.8 × 103 to 7.1 × 107 copies/liter (CV, 0.14). The amount of NoV GII was 104 to 106 copies/liter in July to November, and 106 to 108 copies/liter in December to June, indicating a clear correlation between the amount of NoV GII and the number of outbreaks (Fig. 3). Raw sewage contained a higher amount of NoV GII genome than NoV GI during the survey period.

FIG. 3.

FIG. 3.

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(A) Numbers of outbreaks caused by NoVs in Toyama Prefecture (2006 to 2008). Values are the instances caused by NoV GI.8, GII.4, and GII.13 and instances caused by infection with NoV II agents whose genotypes were not determined, as indicated on the figure. (B) Weekly number of viruses detected from children with gastroenteritis in pediatric clinics and weekly number of patients with gastroenteritis per pediatric clinic in Toyama Prefecture, Japan (2006 to 2008). Data represent patients with NoV GII.4, NoV GII.6, SaV GII.3, and SaV GIV.1, as indicated. The dots indicate the weekly mean number of patients with gastroenteritis from 29 pediatric clinics from April 2006 to March 2008.

NoVs and SaVs detected from clinical cases.

Fifty-nine outbreaks of gastroenteritis occurred, mainly in November to January, during the survey period. Most outbreaks were caused by NoV GII.4, except for two instances caused by GI.8 in April 2006 and GII.13 in December 2007 (Fig. 3A). Genotypes were not determined in two instances. No outbreak caused by SaV occurred.

In 2006 and 2007, the total number of patients with gastroenteritis reported from pediatric clinics sharply increased in early November and then decreased in January (Fig. 3B). Gastroenteritis occurred in the winter season of 2006 to 2007 more frequently than in the season of 2007 to 2008. NoV GII.4 was detected from six patients diagnosed as sporadic gastroenteritis (Fig. 3B). NoV GII.6 and SaV GII.3 and GIV.1 were also observed from one patient each (Fig. 3B). For the other viruses, rotavirus group A (seven children), astrovirus (one child), adenovirus (two children), and parechovirus type 1 (one child) were also detected is samples from children with gastroenteritis (data not shown).

Thus, while NoV GII.4 was the main cause of outbreaks, NoV GII.4 and rotavirus group A were predominant among children with gastroenteritis in Toyama Prefecture from 2006 to 2008.

NoVs and SaVs detected from healthy children.

Since the viruses detected from raw sewage were supposed to be of human origin, we investigated whether they existed in healthy individuals. For this purpose, we examined 134 available stool samples from healthy children and found that NoVs were detected in 17 stools (12.7%) (Table 1). Whereas NoV GII.4 was observed in three samples (2.2%), NoV GII.7 and GII.13 were detected in six (4.5%) and eight (6.0%) samples, respectively. Most NoVs derived from healthy children were observed in January 2007 when NoV GII.4 was prevalent in gastroenteritis cases (Fig. 3). Further investigation throughout the year will be needed to verify the presence of these viruses among overall healthy inhabitants.

TABLE 1.

Viruses detected from feces of healthy children in Toyama, Japan, 2006 to 2008

Date of sample Age of subject (yr) No. of stool samples No. of samples (%) positive fora:
NoV GII.4 NoV GII.7 NoV GII.13
September 2006 <1 12 1
January 2007 <1 2 1
1 10 1 5 2
2 2
3 21
4 0
5 5
6 21 1 6
September 2007 <1 10
January 2008 1 15
2 3
3 9
4 8
5 3
6 13
Total 134 3 (2.2) 6 (4.5) 8 (6.0)
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a

No samples were positive for either NoV GI or SaV.

These results indicate that there were certain healthy children shedding at least NoV GII.4, GII.7, and GII.13 in the winter of 2006 to 2007.

Phylogenetic analysis of NoVs detected from raw sewage and human specimens.

The genetic variations of NoV GI.8 and GII.4, -7, and -13 strains detected from raw sewage were compared with those from human clinical cases by phylogenetic analysis (Fig. 4). NoV GII.4 strains were divided into three clusters: the types of 2006a, 2006b, and Chiba-4e (Fig. 4A) (6, 35, 41, 42). While a strain detected from raw sewage in May 2006 and the strains in July and August 2006 belonged to GII.4 strain 2006a (GII.4/2006a) and GII.4/Chiba-4e, respectively, most of the GII.4 strains from raw sewage belonged to the 2006b cluster. All GII.4 strains derived from patients with gastroenteritis and from healthy children also belonged to the 2006b cluster, except for two GII.4/2006a strains from outbreaks in May 2006 and one GII.4/Chiba-4e strain from a healthy child in September 2006 (Fig. 4B). In addition, the NoV GII.7, GII.13, and GI.8 strains detected from raw sewage formed a cluster with strains detected from healthy children or clinical cases. The identities of nucleotide sequences in 302 bases of the partial capsid regions among these strains were 96.4 to 97.4%, 98.0 to 99.3%, and 99.3 to 100%, respectively (Fig. 4A). Thus, the genotypes of NoVs detected from raw sewage showed a close relationship with those from human cases.

FIG. 4.

FIG. 4.

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Phylogenetic tree for NoV (GII.4, GII.7, GII.13, and GI.8) using about 300 nt from the 3′ end of ORF1 to the beginning of the capsid region; the tree was generated by the neighbor-joining method with Toyama strains derived from raw sewage and reference strains. (A) Phylogenetic tree of the strains of NoV GII.4, GII.7, GII.13, and GI.8. (B) Part of the phylogenetic tree for the strains of NoV GII.4 was magnified with a modification of branch indexes. Filled circles (•) and open squares (□) indicate the Toyama strains derived from raw sewage and asymptomatic healthy children, respectively. Filled triangles (▴) indicate the strains detected from the patients with gastroenteritis in hospitals or outbreaks from April 2006 to March 2008.

DISCUSSION

In this study, we compared NoVs and SaVs detected from raw sewage with those from human specimens. From 2006 to 2008, especially in winter, a large number of sporadic gastroenteritis cases and many outbreaks caused by NoV GII.4 occurred in Toyama, Japan (Fig. 3). NoV GII.4 was also predominantly detected from raw sewage in winter. In addition, the copy number of NoV GII in raw sewage of the winter season of 2006 to 2007 was higher than that of 2007 to 2008 (Fig. 2B), a result that correlates well with the prevalence of gastroenteritis and the number of outbreaks caused by NoV GII.4 infection. Clinical outbreaks preceded the high counts of NoV GII in raw sewage. Phylogenetic analysis showed that the nucleotide sequences of these NoV GII.4 strains were closely related to each other. Therefore, NoVs GII in raw sewage are thought to reflect mainly NoV GII.4 derived from clinical cases.

At least three clusters of NoV GII.4, 2006a, 2006b, and Chiba-4e, appeared to exist in Toyama Prefecture from 2006 to 2008. In Japan including Toyama, NoV GII.4/2006b has been dominantly prevalent since 2006, whereas a few NoV GII.4/2006a strains were detected from patients with gastroenteritis (20, 33). It is uncertain whether NoV GII.4/2006a and GII.4/Chiba-4e were locally extinct or persisted at low levels in Toyama Prefecture. Because NoV GII.4/2006a and GII.4/2006b epidemics had occurred in European countries beginning in December 2005 (22), these three clusters of NoV GII.4 might have migrated from Europe although migration routes have not been clarified.

NoV GI.8 and GII.13 were less frequently observed in outbreaks and were also detected from raw sewage, indicating that raw sewage contained minor genotypes of NoVs in the environment. Because GI.8 was still detected in sewage more than 1 year after the outbreak, GI.8 seemed to circulate over a long period of time in the community.

Various genotypes of NoV (GI.1, -2, -4, -5, -8, -11, -12, and -14; GII.3, -4, -6, -7, -10, -13, -14, and -15) and SaV (GI.1, GI.2, GII.1, and GIV.1) found in raw sewage are predicted to have originated from infected subjects. Among them, NoV GII.7, which was not detected in clinical cases, was found in raw sewage in September 2006 and in six stool specimens of healthy children in January 2007. Moreover, GII.7 and GII.13 were found more frequently than GII.4 in healthy children. These findings suggest that certain NoVs are shed from healthy children and that the population retains these viruses.

Although some other genotypes of NoV and SaV in raw sewage did not correlate with those from clinical cases, our findings suggest that they are also circulating in the environment throughout the year. NoV GI.4 was consistently detected in raw sewage but was not detected from fecal specimens of patients with gastroenteritis or from healthy children. The origin of NoV GI.4 remains to be clarified. Recent work by Okabayashi et al. showed that NoV GII.2, GII.3, GII.8, and GII.12 were detected from asymptomatic food handlers in 2005 and 2006 but not NoV GII.4, despite many outbreaks (34). Healthy adults may be infected with various genotypes of viruses that differ from the prevalent ones causing gastroenteritis. However, these viruses have the potential to be a source of an endemic or epidemic.

In a Mexican study by García et al. (7), nine different genotypes of NoV (GI.1, -3, -5, -7, and -14; GII.1, -2, -7, and -17) were detected in 48 out of 161 stool specimens (29.8%) from asymptomatic children under 2 years of age in June to August 1998. In an Indian study by Monica et al. (31), SaVs (GI.1, -2, and -3; GII.1 and -2) and NoVs (GI.3; GII.2, -3, and -4) were positive in 6 (3.5%) and 7 (4.0%) out of 173 asymptomatic children, respectively, under 3 years old living in an urban slum community in 2001 to 2004. On the other hand, a study in Australia by Marshall et al. (27) showed that NoV was not detected from 399 asymptomatic individuals aged between 5 months and 52 years in July to August 1997. Variation in the detection rate may depend on the differences in sanitation, such as the distribution of the sewage system, age groups of examinees, and methods of viral detection. Generally, improvement of waterworks or sewage facilities is necessary to prevent the transmission of enteric pathogens that infect humans by the fecal-oral route. However, even though the sewage facilities are widely maintained in the Toyama area (86.2% of the population was provided sewage facilities, and most of the rest treated wastewater individually, according to the Toyama Prefectural government in March 2006), the influence of the wastewater that bypasses the treatment system on viral prevalence could not be eliminated. Furthermore, Ueki et al. (44) reported that a few NoV genomes were also detected from treated wastewater of a sewage facility because of the difficulty to inactivate NoV thoroughly by present sewage treatments. The leakage of the NoVs from the sewage treatment system might be an additional cause of the viral prevalence. Continuous existence of NoVs is also probably due to their genetic and antigenic diversity that result from the high mutation rate (9, 25, 41). Another reason seems to be the physical stability of virions in the environment (3, 4, 19) and refractoriness against serum antibodies (2, 15, 38). Moreover, small numbers of NoV virions are reportedly able to establish infection in humans (3, 12), resulting in the easy expansion of viruses in the community. Thus, some genotypes of NoVs may infect healthy children, such as Mexican and Indian children, and outbreaks of gastroenteritis occur every year (7, 31). This study suggests that certain NoVs continuously exist in the community though certain NoVs can become locally extinct. Therefore, surveillance of circulating viruses in the inhabitants is necessary to control and prevent infection by NoVs and SaVs. The above concept correlates with our previous reports showing environmental surveillance of polioviruses and echoviruses that are mostly asymptomatic (14, 30). It is important to inform public health officials about the continuous existence of NoVs and SaVs in the community to prevent outbreaks among inhabitants.

In conclusion, NoVs and SaVs detected from raw sewage reflect their prevalence and circulation in the inhabitants, regardless of symptoms. A combination of the surveys of raw sewage with those of clinical cases helps us to understand the relationship between infection with these viruses and gastroenteritis.

Acknowledgments

This study was supported in part by a Grant for Research on Emerging and Reemerging Infectious Diseases from the Ministry of Health, Labor and Welfare of Japan.

We thank the staffs of hospitals and health centers, those who provided stool specimens, and those who assisted in sampling raw sewage. We are grateful to M. Maekawa for her excellent technical assistance. We thank K. Matsuura for helpful discussion.

Footnotes

▿

Published ahead of print on 5 January 2009.

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