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One-year weekly survey of noroviruses and enteric adenoviruses in the Tone River water in Tokyo metropolitan area, Japan
w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 9 0 5 e2 9 1 0
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
One-year weekly survey of noroviruses and enteric adenoviruses in the Tone River water in Tokyo metropolitan area, Japan Naohiro Kishida a,*, Hisao Morita b, Eiji Haramoto c, Mari Asami a, Michihiro Akiba a a
Division of Water Management, Department of Environmental Health, National Institute of Public Health, 2-3-6 Minami, Wako, Saitama 351-0197, Japan b Saitama Prefectural Water Quality Management Center, 1632 Kobari, Gyoda, Saitama 361-0024, Japan c Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan
article info
abstract
Article history:
To investigate the actual fluctuations in the concentrations of noroviruses (NoVs) GI and
Received 23 August 2011
GII, and enteric adenoviruses (EAdVs) in river water and its relationship with the number of
Received in revised form
acute infectious gastroenteritis patients, one-year weekly quantitative monitoring of NoVs
6 February 2012
GI and GII and EAdVs was performed in the Tone River in Japan where the surface water is
Accepted 4 March 2012
utilized for the main production of drinking water for the Tokyo Metropolitan Area from
Available online 13 March 2012
October 2009 to September 2010. Noroviruses GI and GII and EAdVs were detected in 28 (54%), 33 (63%), and 23 (44%) of the 52 samples (1 L each), respectively. The concentrations
Keywords:
of NoVs GI and GII and EAdVs fluctuated strongly and were more abundant in winter and
Enteric adenoviruses
early spring. The concentration of NoVs GI was transiently greater than 10,000 copies/L.
Gastroenteritis
The number of acute infectious gastroenteritis patients in the upper river basin was highly
Noroviruses
correlated with all the viral concentrations, while general microbial indicator data such as
Real-time PCR
turbidity and heterotrophic plate count were independent of viral concentration as sug-
Waterborne infectious diseases
gested in previous studies. To the best of our knowledge, this is the first study that clearly shows the strong correlation of the number of gastroenteritis with virus contamination in lower river basin. ª 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Noroviruses (NoVs) are the major agents of acute nonbacterial gastroenteritis in patients of all age groups in both developed and developing countries. They are members of the family Caliciviridae and possess a positive-sense polyadenylated single-stranded RNA genome (Green, 2007). 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 (Zheng et al.,
2006). According to the clinical data reported to the National Institute of Infectious Diseases, Japan, GII strains account for the majority (>90%) of acute gastroenteritis cases due to NoVs (National Institute of Infectious Diseases, 2011). A number of waterborne outbreaks of acute gastroenteritis due to NoVs originating from contaminated drinking water and recreational waters have been documented (Nygard et al., 2003; Parshionikar et al., 2003; Hoebe et al., 2004; Maunula et al., 2005). As large quantities of NoVs are shed in the feces of infected patients, NoVs are discharged into the water
* Corresponding author. Tel.: þ81 48 458 6274; fax: þ81 48 458 6275. E-mail address: [email protected] (N. Kishida). 0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2012.03.010
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w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 9 0 5 e2 9 1 0
environment via sewage or night soil treatment systems because it is very difficult to remove NoVs completely in conventional wastewater treatment processes (Haramoto et al., 2006; Kitajima et al., 2009; Nordgren et al., 2009). Once discharged into recipient waters, viruses persist in water environment for long periods depending on temperature and solar irradiation (Allwood et al., 2003). Therefore, NoVs are sometimes detected in the water environment if fecal contamination sources exist (Kitajima et al., 2010). Viral contamination of river water is an important etiological issue, because many rivers receive effluents from wastewater treatment plants upstream and supply drinking water treatment plants downstream. There have been many surveys of NoVs in river water, and NoVs GI and GII were frequently detected (Ueki et al., 2005; Miagostovich et al., 2008; Kitajima et al., 2010; Lodder et al., 2010). However, most studies did not perform frequent repeat surveys. Westrell et al., 2006 indicated that the viral concentrations in river water fluctuated markedly over short periods. Hence, the actual fluctuations in viral concentration have not been determined in sufficient detail. Along with NoVs, other enteric viruses such as enteric adenoviruses (EAdVs) are also important to compare the levels of these viruses, which will also be informative for public health. Human adenoviruses are members of the genus Mastadenovirus in the Adenoviridae family, which comprises 51 serotypes classified into 6 species (AeF). They have double stranded linear DNA and a non-enveloped icosahedral shell with fiber-like projections from each of the 12 vertices (Stewart et al., 1993). Although many EAdVs can replicate in the intestine and are shed in feces, serotypes 40 and 41 of subgroup F are unique in being responsible for most cases of adenovirus-associated gastroenteritis in children. The role of EAdVs in the waterborne outbreak is inconclusive since EAdV infections are mostly asymptomatic among adults due to endemic illness cases and establishment of immunity to this virus (Jiang, 2006). However, EAdVs have been suggested as indicators of viral contamination of human origin because they are often detected in water environment by molecular methods (Albinana-Gimenez et al., 2009). The specific objective of this study was to determine the actual fluctuations in viral concentrations in river water used as a source for drinking water. For this purpose, one-year weekly quantitative monitoring of NoVs GI and GII and EAdVs was performed in the Tone River in Japan, the surface water of which is utilized for the production of drinking water for people living in the Tokyo Metropolitan Area. In addition, the relationships between viral concentration and various indicators such as bacterial concentration and epidemiological data in the upper river basin were investigated.
2.
Materials and methods
2.1. Collection of river water samples and virus concentration A total of 52 river water samples were collected from one sampling site in the Tone River once a week for 1 year, from October 2009 to September 2010. The Tone River has a total
length of 322 km and a catchment area of 16,840 km2, with approximately 800 tributaries. The sampling site was located on the right bank of Tone River at the Tone Diversion Weir, where the surface water is utilized for the production of drinking water for the Tokyo Metropolitan Area. Over 2 million people live in the upper river basin of the sampling site, and there are many wastewater treatment plants and private septic tanks in the area. For each sampling, 1.2 L water sample was collected in a plastic bottle on ice and delivered to the laboratory within 6 h of collection. One liter water was used for viral analysis, while the other 0.2 L water was used for analysis of other items such as turbidity. Bacterial analysis was conducted as soon as the samples arrived. For viral analysis, 1 L water sample was concentrated to approximately 1 mL using electronegative membranes as described previously (Katayama et al., 2002). If the turbidity of the river water was over 3 Nephelometric turbidity unit (NTU), more than 2 electronegative membranes were used. The performance of this concentrating method was validated in our previous studies, and relatively good recovery ratios (15e82%) for NoVs and polioviruses were obtained when it was applied to several kinds of environmental water including river water (Katayama et al., 2008; Haramoto et al., 2009). However, virus concentrations were not compensated using recovery ratios in this study because the ratio of each sample would fluctuate depending on water quality.
2.2.
DNA/RNA extraction and reverse transcription
For detection of EAdVs, DNA extraction was performed as described previously (Haramoto et al., 2007). For the detection of NoVs, extraction of viral RNA and reverse transcription reaction were performed as described previously (Kitajima et al., 2010).
2.3.
Real-time PCR for quantification of viruses
Aliquots of 5 mL of DNA/cDNA sample were mixed with 15 mL of reaction buffer containing 4 mL of 5 LightCycler FastStart DNA MasterPLUS HybProbe Mastermix (Roche Diagnostics, Tokyo, Japan), 400 nM of each primer, and 200 nM of TaqMan probe. The primer pairs and the TaqMan probes used for the detection of EAdVs (Ko et al., 2005) and NoVs GI and GII (Kageyama et al., 2003) were designed to amplify a variety of strains belonging to each virus group. Glass capillaries containing the mixtures were placed into a LightCycler 2.0 (Roche Diagnostics) and incubated at 95 C for 10 min followed by 50 cycles of 95 C for 10 s and 55 C for 30 s for EAdVs, and 95 C for 10 min followed by 50 cycles of 95 C for 10 s and 56 C for 30 s for NoVs. Tenfold serial dilutions of chemically synthesized oligo-DNAs of EAdV serotype 40 (Accession No.: X16583; position: 614e723), NoV GI (Accession No.: M87661; position: 5286e5380), and NoV GII (Accession No.: AF145896; position: 4998e5105) were used to make a standard curve. When fluorescence intensity is over the threshold value within 45 cycles, the sample was considered to be positive. If the detection limit of real-time PCR is 1 copy/reaction, and virus recovery ratio is 50%, the detection limits for NoVs and EAdVs in this study can be calculated to be approximately 350 copies/L and 400 copies/ L, respectively. To improve accuracy of calculating the
2907
6,000 4,000 2,000 0 1000
b 800
3
120 100
600
80 400
60 40
200
20 0
200,000
0
20
c
150,000
15
100,000
10
50,000
5
0
0
Statistical analysis
Spearman’s rank order correlation coefficients (rs) were calculated to evaluate correlations among several indicators and viral concentrations. Analysis was performed using Excel Statistics 2010 (SSRI, Tokyo, Japan). In all analyses, P < 0. 01 was taken to indicate statistical significance. This analysis is often used to evaluate relationships among concentrations of microorganisms (Wilkes et al., 2009). All viral analyses and indicator data obtained in the 1-year survey (n ¼ 52) were used for statistical analyses. If viruses were not detected, one tenth of each calculated detection limit (35 copies/L for NoVs and 40 copies/L for EAdVs) was inputted in this statistical analysis.
3.
Results and discussion
3.1.
Fluctuations in viral concentration in river water
Fig. 1(a) shows the annual profiles of NoVs GI and GII and EAdVs. Noroviruses GI and GII and EAdVs were detected in 28 (54%), 33 (63%), and 23 (44%) of the 52 samples (1 L each), respectively. At least one virus was detected in 40 (77%) of the 52 samples. The time courses of concentrations of NoVs GI and GII in river water were very similar. The concentrations were high in winter and early spring (epidemic season), as expected. There are several peaks of virus concentrations, possibly due to frequent occurrence of several symptomatic and/or asymptomatic infections with enteric viruses in the catchment area. Concentration of NoVs GI was transiently greater than 10,000 copies/L. The EAdVs were also frequently detected with high concentrations in the same season. Although viruses were rarely detected in summer and autumn, NoVs GII were sometimes detected at low concentrations. Meanwhile, virus concentrations of a few water samples in a non-epidemic season were lower than calculated
River flow rate (m /s)
140
ct 1- -09 N ov 1- -09 D ec 1- -09 Ja n 1- -10 Fe b1- 10 M ar 1 - -1 0 A pr 1- -10 M ay 1- -10 Ju n1- 10 Ju l 1- -10 A ug 1- -10 Se p 1- -10 O ct -1 0
2.5.
8,000
Number of acute infectious gastroenteritis patients (persons/surveillance site)
Turbidity and heterotrophic plate count (HPC) were analyzed as indicators of viral concentration in river water according to the Japanese Standard Method for the Examination of Water (Japan Water Works Association, 2001). The numbers of acute infectious gastroenteritis cases (patients/surveillance site) in the upper river basin of the sampling site reported to the local government were also obtained (Gunma Prefectural Institute of Public Health and Environmental Sciences, 2011). In addition, the amount of rainfall and flow rate at the sampling site was measured. These indicator data were obtained once a week for 1 year, from October 2009 to September 2010, along with the viral analysis.
a
10,000
Turbidity (NTU) and amount of rainfall(mm/d)
Indicator data
12,000
1O
2.4.
14,000
160
HPC (MPN/mL)
detection limit, the recovery ratio of each water sample has been obtained by adding surrogates (internal control) such as bacteriophage in recent studies (Rajal et al., 2007a, b). However, surrogates were not added in this study because recovery ratio of each virus is different (Rose et al., 2006; Rigotto et al., 2009), and therefore, it is difficult to compensate the recovery ratio by adding surrogate if more than one virus is targeted.
Virus concentrations (copies/L)
w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 9 0 5 e2 9 1 0
Time
Fig. 1 e Annual profiles of viral concentration and indicator data in the Tone River (a) Noroviruses (NoVs) GI and GII, and enteric adenoviruses (EAdVs). (b) Turbidity, river water flow, and amount of rainfall. (c) Heterotrophic plate count (HPC) and the number of acute infectious gastroenteritis cases. Closed circles: NoVs GI; open circles: NoVs GII; closed triangles: EAdVs; open triangles: turbidity; closed squares: river water flow; vertical bars: amount of rainfall; closed lozenges: HPC; open squares: number of acute infectious gastroenteritis cases.
detection limits. This is because the concentrations were not compensated using recovery ratios of previous studies, and quantified values by real-time PCR strongly fluctuate if the concentration is low. As mentioned above, NoVs GII strains account for the majority of acute gastroenteritis cases due to NoVs in Japan. However, the detection frequency and concentration were not markedly different between the two genogroups. A similar trend was also observed in recent environmental studies of river water samples worldwide (Lee and Kim, 2008; Gentry et al., 2009; Kitajima et al., 2010). These findings suggest that asymptomatic infection with GI strains occurs widely in humans. Moreover, recent studies conducted at wastewater treatment plants have demonstrated that GI has higher persistence following wastewater treatment than GII (Haramoto et al., 2006; Da Silva et al., 2007; Nordgren et al., 2009), suggesting that there may be differences in environmental persistence between NoV strains belonging to GI and GII.
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w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 9 0 5 e2 9 1 0
Table 1 e Spearman’s rank order correlation coefficient matrix among NoVs, EAdVs, and indicator data during 1-year weekly surveys (n [ 52).
a
NoVs GI NoVs GII EAdVsb Turbidity Flow rate Rainfall HPCc Patientsd a b c d e
NoVs GI
NoVs GII
EAdVs
Turbidity
Flow rate
Rainfall
HPC
Patients
1.00 0.72e 0.73e 0.42 0.34 0.12 0.63 0.77e
1.00 0.47e 0.09 0.11 0.08 0.26 0.70e
1.00 0.55 0.51 0.10 0.60 0.52e
1.00 0.83e 0.24 0.74e 0.33
1.00 0.25 0.62e 0.23
1.00 0.28 0.15
1.00 0.51
1.00
Noroviruses. Enteric adenoviruses. Heterotrophic plate count. Number of acute infectious gastroenteritis cases. Significant positive correlation (P < 0.01).
According to limited clinical data reported to the National Institute of Infectious Diseases, Japan, the number of confirmed EAdVs (patients/surveillance site) was large from November to May in 2009/2010 compared with other months (National Institute of Infectious Diseases, 2011). Hence, it is suggested that EAdVs were shed from humans and discharged into the water environment via wastewater treatment plants in this period, which might cause an increase in the concentration of EAdVs in river water. A previous study has also reported that the concentration of EAdVs in a Japanese river increase in winter (Haramoto et al., 2007).
3.2.
Indicators of viral contamination in river water
Fig. 1(b), (c) and Table 1 show the annual profiles of indicator data and the relationships between viral concentration and indicator data, respectively. Significant correlations (P < 0.01) were found among viral concentration with Spearman’s rank order correlation coefficient (rs) ranging from 0.47 to 0.73. The concentration of NoVs GI showed a good correlation with that of NoVs GII. In addition, EAdVs concentration was also strongly correlated with NoVs GI. This would be because the epidemic seasons of the two viruses were almost the same in the 2009/2010 season. The viral concentration was independent of most indicator data such as turbidity and HPC, which have been proposed as possible indicators of contamination by pathogenic microorganisms. The HPC is currently adopted as one of the water quality standard in Japan which indicates bacterial contamination. Several investigators have also suggested that normal indicator data are not always useful in the case of viral contamination in the water environment (Maunula et al., 2005; Westrell et al., 2006). On the other hand, viral contamination was strongly correlated with the number of acute infectious gastroenteritis cases (patients/surveillance site) in the upper river basin of the sampling site reported to the local government. The rs value ranged from 0.52 to 0.77. This is because large quantities of viruses are shed in the feces of infected patients and discharged into the river via wastewater treatment systems. Actually, it has been reported that enteric viruses such as NoVs and EAdVs are discharged from hospital wastewater treatment plant (Prado et al., 2011). Although many previous studies have investigated relationship
between gastroenteritis in the population and virus concentrations or genotypes in sewage wastewater (Iwai et al., 2009; Kamel et al., 2010), the relationship between gastroenteritis and virus concentrations in river water are rarely investigated. To the best of our knowledge, this is the first study that clearly shows the strong correlation of the number of gastroenteritis with virus contamination in lower river basin. Clinical and epidemiological data in the upper river basin are not always available, which decreases the usability of data as indicators. Indeed, we could not obtain sufficient amounts of clinical data of individual viruses for statistical analysis because individual viral analyses were limited at the general local government level. However, nonspecific (macro) data such as the number of acute infectious gastroenteritis cases are readily available in Japan. Hence, it would be possible to use the number of acute infectious gastroenteritis cases as an indicator of viral contamination in many regions.
4.
Conclusions
In this study, 1-year weekly quantitative monitoring of NoVs GI and GII and EAdVs was performed in one Japanese river from which the surface water is utilized to produce drinking water from October 2009 to September 2010. The followings are the main outcomes of this study. (1) Noroviruses GI and GII and EAdVs were detected in 28 (54%), 33 (63%), and 23 (44%) of the 52 samples (1 L each), respectively. The concentrations of NoVs GI and GII and EAdVs fluctuate markedly and these viruses are more abundant in winter and early spring. (2) The number of acute infectious gastroenteritis cases in the upper river basin is clearly correlated with virus concentrations in river water.
Acknowledgment Part of this study was financially supported by the Ministry of Environment, Japan (Environmental Research in Japan, 2009, 4: Evaluation and Control of Health Risk from Human-animal Pollution Sources in Public Water Bodies).
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
One-year weekly survey of noroviruses and enteric adenoviruses in the Tone River water in Tokyo metropolitan area, Japan Naohiro Kishida a,*, Hisao Morita b, Eiji Haramoto c, Mari Asami a, Michihiro Akiba a a
Division of Water Management, Department of Environmental Health, National Institute of Public Health, 2-3-6 Minami, Wako, Saitama 351-0197, Japan b Saitama Prefectural Water Quality Management Center, 1632 Kobari, Gyoda, Saitama 361-0024, Japan c Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan
article info
abstract
Article history:
To investigate the actual fluctuations in the concentrations of noroviruses (NoVs) GI and
Received 23 August 2011
GII, and enteric adenoviruses (EAdVs) in river water and its relationship with the number of
Received in revised form
acute infectious gastroenteritis patients, one-year weekly quantitative monitoring of NoVs
6 February 2012
GI and GII and EAdVs was performed in the Tone River in Japan where the surface water is
Accepted 4 March 2012
utilized for the main production of drinking water for the Tokyo Metropolitan Area from
Available online 13 March 2012
October 2009 to September 2010. Noroviruses GI and GII and EAdVs were detected in 28 (54%), 33 (63%), and 23 (44%) of the 52 samples (1 L each), respectively. The concentrations
Keywords:
of NoVs GI and GII and EAdVs fluctuated strongly and were more abundant in winter and
Enteric adenoviruses
early spring. The concentration of NoVs GI was transiently greater than 10,000 copies/L.
Gastroenteritis
The number of acute infectious gastroenteritis patients in the upper river basin was highly
Noroviruses
correlated with all the viral concentrations, while general microbial indicator data such as
Real-time PCR
turbidity and heterotrophic plate count were independent of viral concentration as sug-
Waterborne infectious diseases
gested in previous studies. To the best of our knowledge, this is the first study that clearly shows the strong correlation of the number of gastroenteritis with virus contamination in lower river basin. ª 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Noroviruses (NoVs) are the major agents of acute nonbacterial gastroenteritis in patients of all age groups in both developed and developing countries. They are members of the family Caliciviridae and possess a positive-sense polyadenylated single-stranded RNA genome (Green, 2007). 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 (Zheng et al.,
2006). According to the clinical data reported to the National Institute of Infectious Diseases, Japan, GII strains account for the majority (>90%) of acute gastroenteritis cases due to NoVs (National Institute of Infectious Diseases, 2011). A number of waterborne outbreaks of acute gastroenteritis due to NoVs originating from contaminated drinking water and recreational waters have been documented (Nygard et al., 2003; Parshionikar et al., 2003; Hoebe et al., 2004; Maunula et al., 2005). As large quantities of NoVs are shed in the feces of infected patients, NoVs are discharged into the water
* Corresponding author. Tel.: þ81 48 458 6274; fax: þ81 48 458 6275. E-mail address: [email protected] (N. Kishida). 0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2012.03.010
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environment via sewage or night soil treatment systems because it is very difficult to remove NoVs completely in conventional wastewater treatment processes (Haramoto et al., 2006; Kitajima et al., 2009; Nordgren et al., 2009). Once discharged into recipient waters, viruses persist in water environment for long periods depending on temperature and solar irradiation (Allwood et al., 2003). Therefore, NoVs are sometimes detected in the water environment if fecal contamination sources exist (Kitajima et al., 2010). Viral contamination of river water is an important etiological issue, because many rivers receive effluents from wastewater treatment plants upstream and supply drinking water treatment plants downstream. There have been many surveys of NoVs in river water, and NoVs GI and GII were frequently detected (Ueki et al., 2005; Miagostovich et al., 2008; Kitajima et al., 2010; Lodder et al., 2010). However, most studies did not perform frequent repeat surveys. Westrell et al., 2006 indicated that the viral concentrations in river water fluctuated markedly over short periods. Hence, the actual fluctuations in viral concentration have not been determined in sufficient detail. Along with NoVs, other enteric viruses such as enteric adenoviruses (EAdVs) are also important to compare the levels of these viruses, which will also be informative for public health. Human adenoviruses are members of the genus Mastadenovirus in the Adenoviridae family, which comprises 51 serotypes classified into 6 species (AeF). They have double stranded linear DNA and a non-enveloped icosahedral shell with fiber-like projections from each of the 12 vertices (Stewart et al., 1993). Although many EAdVs can replicate in the intestine and are shed in feces, serotypes 40 and 41 of subgroup F are unique in being responsible for most cases of adenovirus-associated gastroenteritis in children. The role of EAdVs in the waterborne outbreak is inconclusive since EAdV infections are mostly asymptomatic among adults due to endemic illness cases and establishment of immunity to this virus (Jiang, 2006). However, EAdVs have been suggested as indicators of viral contamination of human origin because they are often detected in water environment by molecular methods (Albinana-Gimenez et al., 2009). The specific objective of this study was to determine the actual fluctuations in viral concentrations in river water used as a source for drinking water. For this purpose, one-year weekly quantitative monitoring of NoVs GI and GII and EAdVs was performed in the Tone River in Japan, the surface water of which is utilized for the production of drinking water for people living in the Tokyo Metropolitan Area. In addition, the relationships between viral concentration and various indicators such as bacterial concentration and epidemiological data in the upper river basin were investigated.
2.
Materials and methods
2.1. Collection of river water samples and virus concentration A total of 52 river water samples were collected from one sampling site in the Tone River once a week for 1 year, from October 2009 to September 2010. The Tone River has a total
length of 322 km and a catchment area of 16,840 km2, with approximately 800 tributaries. The sampling site was located on the right bank of Tone River at the Tone Diversion Weir, where the surface water is utilized for the production of drinking water for the Tokyo Metropolitan Area. Over 2 million people live in the upper river basin of the sampling site, and there are many wastewater treatment plants and private septic tanks in the area. For each sampling, 1.2 L water sample was collected in a plastic bottle on ice and delivered to the laboratory within 6 h of collection. One liter water was used for viral analysis, while the other 0.2 L water was used for analysis of other items such as turbidity. Bacterial analysis was conducted as soon as the samples arrived. For viral analysis, 1 L water sample was concentrated to approximately 1 mL using electronegative membranes as described previously (Katayama et al., 2002). If the turbidity of the river water was over 3 Nephelometric turbidity unit (NTU), more than 2 electronegative membranes were used. The performance of this concentrating method was validated in our previous studies, and relatively good recovery ratios (15e82%) for NoVs and polioviruses were obtained when it was applied to several kinds of environmental water including river water (Katayama et al., 2008; Haramoto et al., 2009). However, virus concentrations were not compensated using recovery ratios in this study because the ratio of each sample would fluctuate depending on water quality.
2.2.
DNA/RNA extraction and reverse transcription
For detection of EAdVs, DNA extraction was performed as described previously (Haramoto et al., 2007). For the detection of NoVs, extraction of viral RNA and reverse transcription reaction were performed as described previously (Kitajima et al., 2010).
2.3.
Real-time PCR for quantification of viruses
Aliquots of 5 mL of DNA/cDNA sample were mixed with 15 mL of reaction buffer containing 4 mL of 5 LightCycler FastStart DNA MasterPLUS HybProbe Mastermix (Roche Diagnostics, Tokyo, Japan), 400 nM of each primer, and 200 nM of TaqMan probe. The primer pairs and the TaqMan probes used for the detection of EAdVs (Ko et al., 2005) and NoVs GI and GII (Kageyama et al., 2003) were designed to amplify a variety of strains belonging to each virus group. Glass capillaries containing the mixtures were placed into a LightCycler 2.0 (Roche Diagnostics) and incubated at 95 C for 10 min followed by 50 cycles of 95 C for 10 s and 55 C for 30 s for EAdVs, and 95 C for 10 min followed by 50 cycles of 95 C for 10 s and 56 C for 30 s for NoVs. Tenfold serial dilutions of chemically synthesized oligo-DNAs of EAdV serotype 40 (Accession No.: X16583; position: 614e723), NoV GI (Accession No.: M87661; position: 5286e5380), and NoV GII (Accession No.: AF145896; position: 4998e5105) were used to make a standard curve. When fluorescence intensity is over the threshold value within 45 cycles, the sample was considered to be positive. If the detection limit of real-time PCR is 1 copy/reaction, and virus recovery ratio is 50%, the detection limits for NoVs and EAdVs in this study can be calculated to be approximately 350 copies/L and 400 copies/ L, respectively. To improve accuracy of calculating the
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6,000 4,000 2,000 0 1000
b 800
3
120 100
600
80 400
60 40
200
20 0
200,000
0
20
c
150,000
15
100,000
10
50,000
5
0
0
Statistical analysis
Spearman’s rank order correlation coefficients (rs) were calculated to evaluate correlations among several indicators and viral concentrations. Analysis was performed using Excel Statistics 2010 (SSRI, Tokyo, Japan). In all analyses, P < 0. 01 was taken to indicate statistical significance. This analysis is often used to evaluate relationships among concentrations of microorganisms (Wilkes et al., 2009). All viral analyses and indicator data obtained in the 1-year survey (n ¼ 52) were used for statistical analyses. If viruses were not detected, one tenth of each calculated detection limit (35 copies/L for NoVs and 40 copies/L for EAdVs) was inputted in this statistical analysis.
3.
Results and discussion
3.1.
Fluctuations in viral concentration in river water
Fig. 1(a) shows the annual profiles of NoVs GI and GII and EAdVs. Noroviruses GI and GII and EAdVs were detected in 28 (54%), 33 (63%), and 23 (44%) of the 52 samples (1 L each), respectively. At least one virus was detected in 40 (77%) of the 52 samples. The time courses of concentrations of NoVs GI and GII in river water were very similar. The concentrations were high in winter and early spring (epidemic season), as expected. There are several peaks of virus concentrations, possibly due to frequent occurrence of several symptomatic and/or asymptomatic infections with enteric viruses in the catchment area. Concentration of NoVs GI was transiently greater than 10,000 copies/L. The EAdVs were also frequently detected with high concentrations in the same season. Although viruses were rarely detected in summer and autumn, NoVs GII were sometimes detected at low concentrations. Meanwhile, virus concentrations of a few water samples in a non-epidemic season were lower than calculated
River flow rate (m /s)
140
ct 1- -09 N ov 1- -09 D ec 1- -09 Ja n 1- -10 Fe b1- 10 M ar 1 - -1 0 A pr 1- -10 M ay 1- -10 Ju n1- 10 Ju l 1- -10 A ug 1- -10 Se p 1- -10 O ct -1 0
2.5.
8,000
Number of acute infectious gastroenteritis patients (persons/surveillance site)
Turbidity and heterotrophic plate count (HPC) were analyzed as indicators of viral concentration in river water according to the Japanese Standard Method for the Examination of Water (Japan Water Works Association, 2001). The numbers of acute infectious gastroenteritis cases (patients/surveillance site) in the upper river basin of the sampling site reported to the local government were also obtained (Gunma Prefectural Institute of Public Health and Environmental Sciences, 2011). In addition, the amount of rainfall and flow rate at the sampling site was measured. These indicator data were obtained once a week for 1 year, from October 2009 to September 2010, along with the viral analysis.
a
10,000
Turbidity (NTU) and amount of rainfall(mm/d)
Indicator data
12,000
1O
2.4.
14,000
160
HPC (MPN/mL)
detection limit, the recovery ratio of each water sample has been obtained by adding surrogates (internal control) such as bacteriophage in recent studies (Rajal et al., 2007a, b). However, surrogates were not added in this study because recovery ratio of each virus is different (Rose et al., 2006; Rigotto et al., 2009), and therefore, it is difficult to compensate the recovery ratio by adding surrogate if more than one virus is targeted.
Virus concentrations (copies/L)
w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 9 0 5 e2 9 1 0
Time
Fig. 1 e Annual profiles of viral concentration and indicator data in the Tone River (a) Noroviruses (NoVs) GI and GII, and enteric adenoviruses (EAdVs). (b) Turbidity, river water flow, and amount of rainfall. (c) Heterotrophic plate count (HPC) and the number of acute infectious gastroenteritis cases. Closed circles: NoVs GI; open circles: NoVs GII; closed triangles: EAdVs; open triangles: turbidity; closed squares: river water flow; vertical bars: amount of rainfall; closed lozenges: HPC; open squares: number of acute infectious gastroenteritis cases.
detection limits. This is because the concentrations were not compensated using recovery ratios of previous studies, and quantified values by real-time PCR strongly fluctuate if the concentration is low. As mentioned above, NoVs GII strains account for the majority of acute gastroenteritis cases due to NoVs in Japan. However, the detection frequency and concentration were not markedly different between the two genogroups. A similar trend was also observed in recent environmental studies of river water samples worldwide (Lee and Kim, 2008; Gentry et al., 2009; Kitajima et al., 2010). These findings suggest that asymptomatic infection with GI strains occurs widely in humans. Moreover, recent studies conducted at wastewater treatment plants have demonstrated that GI has higher persistence following wastewater treatment than GII (Haramoto et al., 2006; Da Silva et al., 2007; Nordgren et al., 2009), suggesting that there may be differences in environmental persistence between NoV strains belonging to GI and GII.
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Table 1 e Spearman’s rank order correlation coefficient matrix among NoVs, EAdVs, and indicator data during 1-year weekly surveys (n [ 52).
a
NoVs GI NoVs GII EAdVsb Turbidity Flow rate Rainfall HPCc Patientsd a b c d e
NoVs GI
NoVs GII
EAdVs
Turbidity
Flow rate
Rainfall
HPC
Patients
1.00 0.72e 0.73e 0.42 0.34 0.12 0.63 0.77e
1.00 0.47e 0.09 0.11 0.08 0.26 0.70e
1.00 0.55 0.51 0.10 0.60 0.52e
1.00 0.83e 0.24 0.74e 0.33
1.00 0.25 0.62e 0.23
1.00 0.28 0.15
1.00 0.51
1.00
Noroviruses. Enteric adenoviruses. Heterotrophic plate count. Number of acute infectious gastroenteritis cases. Significant positive correlation (P < 0.01).
According to limited clinical data reported to the National Institute of Infectious Diseases, Japan, the number of confirmed EAdVs (patients/surveillance site) was large from November to May in 2009/2010 compared with other months (National Institute of Infectious Diseases, 2011). Hence, it is suggested that EAdVs were shed from humans and discharged into the water environment via wastewater treatment plants in this period, which might cause an increase in the concentration of EAdVs in river water. A previous study has also reported that the concentration of EAdVs in a Japanese river increase in winter (Haramoto et al., 2007).
3.2.
Indicators of viral contamination in river water
Fig. 1(b), (c) and Table 1 show the annual profiles of indicator data and the relationships between viral concentration and indicator data, respectively. Significant correlations (P < 0.01) were found among viral concentration with Spearman’s rank order correlation coefficient (rs) ranging from 0.47 to 0.73. The concentration of NoVs GI showed a good correlation with that of NoVs GII. In addition, EAdVs concentration was also strongly correlated with NoVs GI. This would be because the epidemic seasons of the two viruses were almost the same in the 2009/2010 season. The viral concentration was independent of most indicator data such as turbidity and HPC, which have been proposed as possible indicators of contamination by pathogenic microorganisms. The HPC is currently adopted as one of the water quality standard in Japan which indicates bacterial contamination. Several investigators have also suggested that normal indicator data are not always useful in the case of viral contamination in the water environment (Maunula et al., 2005; Westrell et al., 2006). On the other hand, viral contamination was strongly correlated with the number of acute infectious gastroenteritis cases (patients/surveillance site) in the upper river basin of the sampling site reported to the local government. The rs value ranged from 0.52 to 0.77. This is because large quantities of viruses are shed in the feces of infected patients and discharged into the river via wastewater treatment systems. Actually, it has been reported that enteric viruses such as NoVs and EAdVs are discharged from hospital wastewater treatment plant (Prado et al., 2011). Although many previous studies have investigated relationship
between gastroenteritis in the population and virus concentrations or genotypes in sewage wastewater (Iwai et al., 2009; Kamel et al., 2010), the relationship between gastroenteritis and virus concentrations in river water are rarely investigated. To the best of our knowledge, this is the first study that clearly shows the strong correlation of the number of gastroenteritis with virus contamination in lower river basin. Clinical and epidemiological data in the upper river basin are not always available, which decreases the usability of data as indicators. Indeed, we could not obtain sufficient amounts of clinical data of individual viruses for statistical analysis because individual viral analyses were limited at the general local government level. However, nonspecific (macro) data such as the number of acute infectious gastroenteritis cases are readily available in Japan. Hence, it would be possible to use the number of acute infectious gastroenteritis cases as an indicator of viral contamination in many regions.
4.
Conclusions
In this study, 1-year weekly quantitative monitoring of NoVs GI and GII and EAdVs was performed in one Japanese river from which the surface water is utilized to produce drinking water from October 2009 to September 2010. The followings are the main outcomes of this study. (1) Noroviruses GI and GII and EAdVs were detected in 28 (54%), 33 (63%), and 23 (44%) of the 52 samples (1 L each), respectively. The concentrations of NoVs GI and GII and EAdVs fluctuate markedly and these viruses are more abundant in winter and early spring. (2) The number of acute infectious gastroenteritis cases in the upper river basin is clearly correlated with virus concentrations in river water.
Acknowledgment Part of this study was financially supported by the Ministry of Environment, Japan (Environmental Research in Japan, 2009, 4: Evaluation and Control of Health Risk from Human-animal Pollution Sources in Public Water Bodies).
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