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We investigated the prevalence, seasonal dependence, and genetic diversity of noroviruses (NoVs) in the Tamagawa River, which runs through a densely populated region in Tokyo, Japan, from April 2003 to March 2004. A total of 60 water samples were collected from five sites from upstream to downstream along the river, and 500 mL of which was concentrated using the “cation-coated filter method”. Of the 60 samples tested, genogroup I (GI) and GII NoVs were detected from 28 (47%) and 18 (30%) samples, respectively. GIV NoV was successfully detected from 2 (3%) samples with a newly developed seminested RT-PCR assay specific for GIV. The occurrence of NoVs in the river was significantly higher in winter/spring than in summer/autumn and also in mid- to downstream rather than upstream. A total of 176 different NoV strains were identified from river water samples based on the phylogenetic analysis of partial capsid gene sequences. GI, GII, and GIV strains were clustered into 7 (GI/1, 2, 4, 5, 8, 9, and 11), 8 (GII/ 2, 3, 4, 5, 6, 11, 12, and 16), and 1 (GIV/1) genotypes, respectively. The results suggest that genetically diverse NoV strains are circulating between human populations and aquatic environments.
NoVs show high genetic diversity and are currently proposed to be divided into five genetically distinct genogroups, genogroups I (GI) to V (GV), of which GI, GII, and GIV infect humans. The strains in GI and GII can be further subdivided into genotypes (2). Human NoVs infect all ages and cause various symptoms such as watery nonbloody diarrhea, vomiting, abdominal cramps, and nausea (1). According to the clinical data reported to the National Institute of Infectious Diseases, Japan (3), GII strains account for the majority (more than 90%) of acute gastroenteritis cases due to NoVs, whereas GI strains comprise of the remaining cases (up to 10%). Compared with GI and GII NoVs, GIV NoV has seldom been detected in fecal samples from gastroenteritis patients, although it is also likely to cause acute gastroenteritis (4, 5). In Japan, the occurrence of gastroenteritis illnesses due to NoVs shows a clear seasonal trend, with outbreaks peaking in the winter/spring period, from November to April (3). In addition, NoVs are considered to be extremely infectious (6), and a number of waterborne outbreaks of acute gastroenteritis due to NoVs originating from contaminated drinking water and recreational waters have been documented (7-10). Since large quantities of NoVs are shed in the feces of infected patients, raw sewage contains the viruses in high concentration, especially during the epidemic season (11-16). Our previous studies have demonstrated that it is very difficult to achieve complete removal of NoVs with conventional wastewater treatment processes, resulting in frequent detection of the viruses even in treated sewage (11-13). Thus, NoVs are abundant in environmental settings, such as river water, seawater, and shellfish, even in areas with proper coverage of wastewater treatment plants (WWTPs). NoVs are included in the latest U.S. Environmental Protection Agency’s Contaminant Candidate List (CCL 3), a list of emerging contaminants that may pose a public health risk in aquatic environments (17). Viral contamination of river water is an important etiological issue, because there are many rivers that receive effluents from WWTPs upstream and supply to drinking water treatment plants (DWTPs) downstream. In other words, monitoring urban river water could be an appropriate approach in understanding the real prevalence of viruses in the catchment areas, because urban rivers receive effluents from multiple WWTPs containing viruses shed from both symptomatic and asymptomatic individuals in the catchment areas. We selected the Tamagawa River in Japan as a typical example of such rivers. This study was performed on the basis of the above background, to investigate the prevalence, seasonal dependence, and genetic diversity of NoVs of all human genogroups in the river, during a one year period. NoV genomes in the river water were detected with seminested RT-PCR assays, including a newly developed assay for GIV NoV, and the strains were further characterized based on partial capsid gene sequences.
Introduction
Materials and Methods
Seasonal Distribution and Genetic Diversity of Genogroups I, II, and IV Noroviruses in the Tamagawa River, Japan M A S A A K I K I T A J I M A , * ,† TOMOICHIRO OKA,‡ EIJI HARAMOTO,§ NAOKAZU TAKEDA,| KAZUHIKO KATAYAMA,‡ AND HIROYUKI KATAYAMA† Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan, and Research Collaboration Center on Emerging and Re-emerging Infections, National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Tivanond 14 Road, Muang, Nonthaburi 11000, Thailand
Received February 3, 2010. Revised manuscript received July 20, 2010. Accepted July 26, 2010.
Noroviruses (NoVs) are a major cause of gastroenteritis worldwide. They are members of the family Caliciviridae and possess a positive-sense polyadenylated single-stranded RNA genome with three open reading frames (ORFs) (1). * Corresponding author phone: +81-3-5841-6242; fax: +81-3-58416244; e-mail:
[email protected]. † The University of Tokyo. ‡ National Institute of Infectious Diseases. § University of Yamanashi. | Ministry of Public Health. 7116
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Collection and Concentration of River Water Samples. In order to detect human NoV genomes in river water, a total of 60 samples were collected monthly from April 2003 to March 2004 at five sites (sites 1 to 5 from the upstream side) along the Tamagawa River in Japan (see Figure S1, Supporting Information). All samples were collected on sunny or cloudy days in plastic bottles, stored on ice, and delivered to the laboratory. Detailed information on the Tamagawa River is indicated in the Supporting Information. The viruses in the river water samples were concentrated within 12 h after collection using a method termed “cation10.1021/es100346a
2010 American Chemical Society
Published on Web 08/18/2010
TABLE 1. Primers Used in Seminested RT-PCR To Amplify the Human Norovirus Capsid N/S Region genogroup
PCR
primer
sequence (5′-3′)a
polarityb
location
reference
GI GI GI
first first and second second
COG1F G1SKR G1SKF
CGYTGGATGCGNTTYCATGA CCAACCCARCCATTRTACA CTGCCCGAATTYGTAAATGA
+ +
5291-5310 5653-5671c 5342-5362c
21 22 22
GII GII GII
first first and second second
COG2F G2SKR G2SKF
CARGARBCNATGTTYAGRTGGATGAG CCRCCNGCATRHCCRTTRTACAT CNTGGGAGGGCGATCGCAA
+ +
5003-5028d 5379-5401d 5058-5076d
21 22 22
GIV GIV GIV
first first and second second
COG4F G4SKR G4SKF
TTTGAGTCYATGTAYAAGTGGATGC CCWCCAGCATATGARTTGTACAT CTTGGGAGGGGGATCGCGA
+ +
686-710e 1050-1072e 729-747e
this study this study this study
c
a Mixed bases in degenerate primers are as follows: Y stands for C or T; N stands for A, C, G, or T; R stands for A or G; B stands for G, T, or C; H stands for A, C, or T. b +, forward primer; -, reverse primer. c Corresponding nucleotide position of Norwalk/68/US (accession no. M87661). d Corresponding nucleotide position of Lordsdale/93/UK (accession no. X86557). e Corresponding nucleotide position of Saint Cloud/98/US (accession no. AF414427).
coated filter method”, as previously described (18). Detailed protocols of virus concentration are also indicated in the Supporting Information. Primer Design for GIV NoV Detection. Currently, only several partial genome sequences of GIV NoVs are available from public databases (4, 5, 19, 20). Seminested PCR primers for detection of GIV NoVs were newly designed to target the conserved region located in the 5′ terminus of ORF2, to amplify all currently known human GIV NoV strains (see Figure S2, Supporting Information). The strains and accession numbers of human GIV NoVs used for primer design were as follows: Saint Cloud (AF414427), Fort Lauderdale (AF414426), Italy (FM865412), Alphatron (AF195847), and Alph23 (DQ093067). The reverse primer (G4SKR) was designed based on the five strains, and the forward primers (COG4F and G4SKF) were designed based on three strains because Alphatron and Alph23 strains lack the sequences in this region (see Figure S2, Supporting Information). The newly designed primers, COG4F, G4SKF, and G4SKR (shown in Table 1), generates 344 bp products by seminested PCR, corresponding to the partial human GIV NoV genome nucleotide position of 7291072 (Saint Cloud strain; Table 1), which is located in the 3′ terminus of ORF1 and 5′ terminus of ORF2. RNA Extraction and Seminested RT-PCR. Viral RNA was extracted from 140 µL of concentrated sample using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) to obtain a final volume of 60 µL according to the manufacturer’s protocol. The RT reaction was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The first PCR was performed with COG1F and G1SKR primers for GI, COG2F and G2SKR primers for GII, and COG4F and G4SKR primers for GIV to generate approximately 380, 390, and 390 bp products, respectively. Subsequently, the second PCR was performed with G1SKF and G1SKR primers for GI, G2SKF, and G2SKR primers for GII, and G4SKF and G4SKR primers for GIV to generate approximately 330, 340, and 340 bp products, respectively. Primers for GI and GII NoVs are described previously (21, 22; Table 1), and detailed seminested RT-PCR protocols are indicated in the Supporting Information. Cloning, Sequencing, and Phylogenetic Analysis. The second PCR products were separated by electrophoresis on 2% agarose gel and visualized under a UV lamp following ethidium bromide staining. PCR products of expected size were excised from the gel and purified using the QIAquick Gel Extraction Kit (Qiagen). Cloning, sequencing, and phylogenetic analysis were performed as described in the Supporting Information.
The nucleotide sequences determined in this study have been deposited in GenBank under accession numbers AB536978 to AB537160. Statistics. χ2 tests were performed to determine whether there is a significant difference among seasons and sampling sites.
Results Detection of NoVs in River Water Samples. A total of 60 water samples (500 mL each) were collected from the Tamagawa River and examined for NoVs; the results are summarized in Table 2. Of the 60 samples, GI and GII NoVs were detected from 28 (47%) and 18 (30%) samples, respectively, by seminested RT-PCR assays using the primers as described previously (21, 22; Table 1). GIV NoV was successfully detected from 2 (3%) samples collected at site 3 in April 2003 and at site 4 in March 2004 with a seminested RT-PCR assay using the newly designed primers (Table 1). At least one genogroup was detected in 30 (50%) samples. The coexistence of GI and GII NoVs was identified in 16 (27%) samples and that of GI and GIV NoVs was also identified in 2 (3%) samples. Meanwhile, coexistence of GII and GIV NoVs was not observed, and, consequently, coexistence of three genogroups was also not observed. Seasonal and Spatial Distribution of NoVs in the Tamagawa River. In order to investigate differences in seasons and spatial difference, we collected samples from five sites along the river (Figure S1) monthly, from April 2003 to March 2004. NoVs of any genogroup were detected at 4 (80%) of five sites in April 2003, in 2 (40%) sites in June 2003, and more than 3 (60%) sites from October 2003 to March 2004, whereas no NoVs were detected either in May 2003 or from July to September 2003 (Table 2). There was a significant difference between the positive ratio of NoVs in winter/spring (April 2003 and from October 2003 to March 2004) and summer/autumn (May 2003 to September 2003) (χ2 test; P < 0.05), demonstrating higher NoV occurrence in the river in the winter/spring season. At the sampling sites, NoV was detected in only 2 (17%) out of 12 months in the upstream area (site 1), whereas in more than 6 (50%) out of 12 months in the mid- to downstream area (sites 2 to 5) (Table 2). There was also a significant difference between the positive ratio of NoVs in upstream and mid- to downstream areas (χ2 test; P < 0.05), demonstrating higher NoV occurrence in the downstream area. Genetic Diversity of Human NoVs Detected in River Water Samples. To investigate the genetic diversity of NoV strains in the Tamagawa River, the nucleotide sequences of the cloned PCR products (eight clones for each genogroups per sample) were aligned, and a phylogenetic dendrogram was constructed based on partial capsid gene sequences VOL. 44, NO. 18, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Detection of Noroviruses in the Tamagawa River, Japan seminested RT-PCRa year/month
site 1
2003/Apr May June July Aug Sep Oct Nov Dec 2004/Jan Feb Mar
-b GII GII
site 2 GI GI GI GI GI GI GI
+ + + + +
GII GII GII GII GII
site 3 GI GI GI GI GI GI GI GI
+ GIV
+ + + + +
GII GII GII GII GII
site 4 GI GI GI + GII GI GI + GII GI GI + GIV
site 5 GI GI GI GI GI GI
+ GII + GII + GII + GII
genotypes identified GI/1, GI/4 GI/4, GI/1, GI/2, GI/1, GI/1, GI/1,
GI/2, GI/4, GIV/1
GI/9 GI/2, GI/4, GI/4, GI/2, GI/4,
GI/4, GI/8, GI/11, GII/3, GII/4, GII/6 GI/5, GII/3, GII/4, GII/6 GI/5, GII/2, GII/3, GII/4, GII/5, GII/6, GII/16 GI/4, GII/3, GII/4, GII/6, GII/11, GII12 GII/4, GII/5, GII/6, GIV/1
a Significant difference between the positive ratio in winter/spring (April 2003 and from October 2003 to March 2004) and summer/autumn (May 2003 to September 2003) (χ2 test; P < 0.05), in upstream (site 1) and mid- to downstream areas (sites 2 to 5) (χ2 test; P < 0.05). b -, not detected.
(approximately 290, 280, and 280 bp for GI, GII, and GIV, respectively; Figures 1-3). Phylogenetic analysis revealed genetic diversity and existence for a large number of different NoV strains in the river water. We identified a total of 176 different NoV strains from the river water, comprising 110 GI, 64 GII, and 2 GIV strains. GI strains were classified into 7 genotypes, namely GI/1, 2, 4, 5, 8, 9, and 11. The major GI genotypes in the present study were GI/1 and GI/4, which were identified in 14 (23%) and 23 (38%) of 60 samples, respectively. GII NoV strains were classified into 8 genotypes, namely GII/2, 3, 4, 5, 6, 11, 12, and 16. The major GII genotypes were GII/3, GII/4, and GII/6, which were identified in 7 (12%), 9 (15%), and 11 (18%) of 60 samples, respectively. Two GIV/1 strains were identified in this study (Figure 3), and their sequences were closely matched (99.3-99.6%) to that of Alph23 strain (DQ093067) identified from viral gastroenteritis patients in Japan (19). A total of 16 different genotypes of NoV were identified in this study. During the period of investigation, the highest number of genotypes (9 genotypes) was identified in January 2004, demonstrating the coexistence of diverse genotypes of NoV strains in the river water and occurrence of NoV infections due to multiple genotypes in the catchment area during this season.
Discussion NoVs are the most significant viral pathogen associated with foodborne and waterborne outbreaks of acute gastroenteritis, worldwide (1). Environmental contamination by NoVs is well documented (18, 23-29), and thus outbreaks due to contaminated water and food pose a serious health risk to humans. In other words, circulation of NoVs between contaminated environmental water and human populations is a key issue in understanding their epidemiology and health risks for humans. In the present study, we investigated the prevalence, seasonal dependence, and genetic diversity of GI, GII, and GIV NoVs in the Tamagawa River in Japan. The presence of NoV genomes in the samples was examined using seminested RT-PCR assays, including a newly developed assay for GIV NoVs, targeting the 5′ terminus of the capsid gene region. We collected water samples at five sites along the Tamagawa River to identify the differences in the occurrence of human NoVs among the sampling sites. NoV sequence was rarely detected in the upstream area (site 1), whereas sampling sites 2, 3, and 4 showed high positive rates (Table 2). This is mainly because midstream and downstream areas of the river are highly contaminated with effluents from several upstream WWTPs as well as effluents from septic tanks installed in the upstream area (Figure S1). The 7118
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introduction of advanced wastewater treatment process, such as ozonation or UV disinfection, will contribute to achieving a microbiologically safe quality of river water. During a one year investigation, NoVs were frequently detected in the winter/ spring season (Table 2). The number of gastroenteritis patients with NoV infection increases in winter in Japan. Moreover, our previous studies demonstrated that higher NoV concentrations were observed not only in influents but also in effluents of WWTPs during the winter season in Japan (11-13), suggesting that higher quantities of NoVs were discharged into the rivers from WWTPs in winter. Conversely, the concentration of NoVs in wastewater was significantly lower in summer (i.e., nonepidemic period of NoVs) than in winter (11-13). A previous study investigated the occurrence of NoVs in river water for a two year period and reported that NoVs were frequently identified in the winter season (30), which is consistent with the results obtained in the present study. We previously examined these river water samples for NoVs with TaqMan-based quantitative real-time RT-PCR assays (13, 18), and NoVs were detected in higher concentrations in winter than in summer, demonstrating results consistent with the present study. In these previous studies, we identified NoV genomes in river water quantitatively but were unable to confirm whether they were derived from viable NoV particles. However, the results described in these studies may provide useful information in understanding the occurrence of NoVs in river water. Recently, quantitative approaches to understanding NoV infection risks have been documented (6, 31). Further analytical study should be conducted to characterize the potential risk of infection when river water is used for drinking water sources or recreational purposes with quantitative microbial risk assessment framework. It should be noted that GI NoV was more frequently detected than GII NoV. This result disagrees with the epidemiological data obtained during the period 2003-2004 from the Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Japan, where GII strains were much more frequently detected than GI in feces of hospitalized patients (3). In addition, a number of clinical studies in Japan also reported GII strains as being more predominantly associated with viral gastroenteritis outbreaks due to NoVs (16, 27, 32). However, in contrast, results similar to those of the present study have been obtained in recent environmental studies of both river water samples in Korea and estuarine samples in the U.S (23, 26). Our previous studies also demonstrated that not only GII but also GI NoVs can be identified in raw sewage, throughout the year (11, 12). These findings suggest that GI strains are more widely spread among humans than previously thought. Moreover, recent studies
FIGURE 1. Phylogenetic tree for GI NoV strains using about 290 nt of partial capsid gene sequences; the tree was generated by the neighbor-joining method, with the strains derived from river water and reference strains. (a) Phylogenetic tree of GI strains. GI/1, GI/ 2, GI/4, GI/5, GI/8, GI/9, and GI/11 represent genotypes identified in this study. Part of the phylogenetic tree for the strains of (b) GI/1 and (c) GI/4 was magnified. The numbers on each branch indicate the bootstrap values for the genotype, and bootstrap values of 950 or higher were considered statistically significant for the grouping. The scale represents nucleotide substitutions per site. Strains shown in italic bold are NoVs detected in this study, representing the year, month, and sampling site. conducted at full-scale WWTPs in Japan, France, and Sweden have demonstrated that GI generally has higher persistence against wastewater treatment than GII (11, 14, 33), suggesting that there may be differences in environmental persistence between NoV strains belonging to GI and GII. Behavior of GIV strains in aquatic environments remains unknown, since there have been only a few studies reporting detection of GIV NoV in environmental samples (5, 15). Our recent report described quantitative data on the occurrence of GIV NoV in wastewater and river water over a one year period (13), which may provide useful data toward understanding its occurrence and fate in aquatic environments. In the present study, we developed a seminested RT-PCR assay to amplify the partial capsid gene of GIV NoV and successfully identified 2 strains in the river water (Figure 3). This assay is expected
to be a useful tool in identifying GIV strains not only from environmental samples but also from clinical specimens. NoV strains detected in river water were genetically diverse, and a total of 16 different genotypes were identified in the water samples, namely GI/1, GI/2, GI/4, GI/5, GI/8, GI/9, GI/11, GII/2, GII/3, GII/4, GII/5, GII/6, GII/11, GII/12, GII/16, and GIV/1 (Table 2, Figures 1-3). Among these genotypes, GI/4 was detected at an especially high rate, being present in 23 (38%) water samples, and a total of 71 GI/4 strains were identified (Figure 1c). Iwai et al. (16) also reported that GI/4 was consistently detected in raw sewage collected from 2006 to 2008; however, it was detected in fecal specimens from neither gastroenteritis patients nor healthy children. Recent clinical studies on NoVs have indicated that there are asymptomatic people with high viral loads (34) and that VOL. 44, NO. 18, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Phylogenetic tree for GII NoV strains using about 280 nt of partial capsid gene sequences; the tree was generated by the neighbor-joining method, with the strains derived from river water and reference strains. (a) Phylogenetic tree of GII strains. GII/2, GII/3, GII/4, GII/5, GII/6, GII/11, GII/12, and GII/16 represent genotypes identified in this study. Part of the phylogenetic tree for the strains of (b) GII/4 and (c) GII/6 was magnified. The numbers on each branch indicate the bootstrap values for the genotype, and bootstrap values of 950 or higher were considered statistically significant for the grouping. The scale represents nucleotide substitutions per site. Strains shown in italic bold are NoVs detected in this study, representing the year, month, and sampling site.
FIGURE 3. Phylogenetic tree for GIV NoV strains using about 280 nt of partial capsid gene sequences; the tree was generated by the neighbor-joining method, with the strains derived from river water and reference strains. The numbers on each branch indicate the bootstrap values for the genotype, and bootstrap values of 950 or higher were considered statistically significant for the grouping. The scale represents nucleotide substitutions per site. Strains shown in italic bold are NoVs detected in this study, representing the year, month, and sampling site. prolonged NoV excretion is observed even after recovery from gastroenteritis symptoms (35-39). However, the relationship between genotypes and their pathogenicity or symptoms has not been well characterized. NoV strains detected in the environment may reflect, more accurately, the actual circulation in the population rather than reported cases which represent a small proportion of total cases. Monitoring of river water could prove to be an appropriate approach in understanding the real prevalence of the viruses in the river catchment areas, because most urban river water receives 7120
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effluents from multiple WWTPs that contain viruses shed from all patients in the catchment areas. The Tamagawa River runs through a densely populated region in Tokyo, Japan, and receives effluents from several WWTPs in the catchment area. This study demonstrates that genetically diverse NoV strains, including GIV, are circulating between human populations and aquatic environments. In conclusion, this study describes novel findings on the prevalence, seasonal dependence, and genetic diversity of NoV strains, including GIV, in river water. Furthermore, this study highlights the importance of further environmental studies, even in combination with clinical studies toward a better understanding of the circulation of NoVs in aquatic environments and human populations.
Acknowledgments This work was supported by Grant-in-Aid for Young Scientists (A) under project number 20686035 from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a grant from the Ministry of Health, Labour, and Welfare of Japan.
Supporting Information Available Additional details on Materials and Methods (Table S1 and Figures S1 and S2). This material is available free of charge via the Internet at http://pubs.acs.org.
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