Environ. Sci. Technol. 2006, 40, 7423-7427
Genetic Variation in the Conservative Gene Region of Norovirus Genogroup II Strains in Environmental and Stool Samples† D A I S U K E S A N O , * ,‡ Y O U U E K I , § TORU WATANABE,‡ AND TATSUO OMURA‡ Department of Civil Engineering, Graduate School of Engineering, Tohoku University, Aoba 6-6-06, Sendai 980-8579, Japan, and Miyagi Prefectural Institute of Public Health and Environment, Saiwai-cho 4-7-2, Miyagino-ku, Sendai 983-0836, Japan
Noroviruses (NoVs) have been one of leading etiological agents for infectious gastroenteritis over the world. Gastroenteritis caused by NoVs is prevalent in winter season, and the contamination of the water environment with NoVs in the epidemic cold season is frequently reported. In contrast, the number of gastroenteritis patients and NoVs in the water environment are reduced during the nonepidemic summer season, and the year-round fate of NoVs has remained to be elucidated. In this study, we collected nucleotide sequences of NoV genogroup II (GII) from domestic sewage, sewage sludge, treated wastewater, river water, and stool samples of gastroenteritis patients in geographically close areas. Phylogenetic analysis of the obtained NoV gene revealed that six out of seven isolates from environmental samples and 10 out of 11 isolates from stool samples belong to genotype 3 (NoV GII.3) or 4 (NoV GII.4), which have been prevalent throughout the world. Genetic distances between the conservative gene region of NoV GII.4 variants implied that genetically diverse strains are likely to occur in environmental samples. The evaluation of the evolutionary change of NoV gene obtained from environmental samples would make it possible to elucidate the year-round fate of NoVs.
Introduction Noroviruses (NoVs), previously called Norwalk-like viruses or small round structured viruses, have been leading etiological agents of acute gastroenteritis all over the world (1). NoVs are in the genus Norovirus within the family Caliciviridae, and the genus Norovirus is divided into five genogroups GI, GII, GIII, GIV, and GV. Strains in each genogroup can be further divided into genotypes (2). These viruses infect humans of all ages and cause symptoms of nausea, vomiting, diarrhea, abdominal cramps, headache, fever, and so on (3). The number of gastroenteritis cases caused by NoVs are growing, and the annual number of NoV isolates from gastroenteritis patients in Japan reached 2600 in 2005, which increased about 10-fold during this decade. The epidemic †
This article is part of the Emerging Contaminants Special Issue. * Corresponding author phone: +81-22-795-7483; fax: +81-22795-7482; e-mail:
[email protected]. ‡ Tohoku University. § Miyagi Prefectural Institute of Public Health and Environment. 10.1021/es0603453 CCC: $33.50 Published on Web 08/15/2006
2006 American Chemical Society
season of the infectious gastroenteritis caused by NoVs ranges from November to April in Japan, in which the monthly average number of food-poisoning incidences by NoVs for the past 5 years is 33 cases. Since NoVs are excreted in stools of gastroenteritis patients, domestic sewage includes a large amount of NoVs in the epidemic season (4, 5). There are severe difficulties in removing and inactivating NoVs in conventional wastewater treatment processes (6), so contaminations of the water environment with NoVs in the epidemic season are frequently reported (2, 5, 6, 7). In contrast, the number of gastroenteritis patients and NoVs in the water environment are significantly reduced during the non-epidemic season (between May and October in Japan), in which the monthly average number of food-poisoning incidences caused by NoVs for the past 5 years is 4 cases. Although sporadic occurrences of NoVs in water (7-9), shellfish (10), and stool samples from gastroenteritis patients (11) have been observed even in the non-epidemic season, the year-round fate of NoVs remains to be elucidated. In this study, nucleotide sequences of the NoV GII gene were collected from environmental (domestic sewage, sewage sludge, treated wastewater, and river water) and stool samples in geographically close areas, in which we have already confirmed the frequent occurrence of NoV GII strains in the winter season (5). NoV GII strains cause acute gastroenteritis more frequently than NoV GI strains not only in Japan (12) but also in other countries (13). The nucleotide sequences of the NoV GII gene obtained in the area under investigation were used for phylogenetic anlaysis, and the fate of the NoV GII strains was discussed based on the genetic distances with the conserved gene region of the NoV GII strains.
Experimental Section Environmental and Stool Samples. The research area is in Miyagi prefecture in the northeast of Japan (Figure 1). The Takagi River runs through Matsushima and flows into Matsushima Bay. There is only one wastewater treatment plant (WWTP) along the Takagi River, which employs the oxidation ditch system for the treatment of about 4000 m3 per day of domestic wastewater from residences in Matsushima. The treated wastewater is chlorinated for disinfection and discharged into the Takagi River. NoV genes in river water from the Takagi River were investigated with reverse transcription polymerase chain reaction (RT-PCR) once a month in July, August, and September of 2004 and July and August of 2005. Sampling points are indicated with black circles in Figure 1. Since treated wastewater from the WWTP was considered to be one of the main contamination sources of NoVs in this area (5), samples of domestic wastewater, sewage sludge and treated wastewater from the WWTP were collected at the same sampling date. Stool samples of gastroenteritis patients were also collected from hospitals in geographically close areas between April and September of 2004 and 2005. Concentrations of NoVs in Environmental and Stool Samples. NoVs in 1 L of river water and treated wastewater were concentrated by polyethylene glycol precipitation according to Lewis and Metcalf (14). The pellet was suspended in 2 mL of distilled deionized water (DDW, Nippon Gene, Tokyo, Japan). NoVs in domestic wastewater and sewage sludge were recovered as described previously (15). First, samples were centrifuged at 9000g for 15 min at 4 °C, and NoVs in the supernatant were concentrated by polyethylene glycol precipitation. NoVs in the pellet were eluted by the EVE method (15), in which the pellet was suspended in 400 mL of EVE buffer containing 1% lysozyme and 0.1 mequiv VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Source, Positive Rate, Serial Number, Genotype, and Collection Date of the Norovirus GII Gene source domestic wastewater
positive ratea 60 (3/5)
serial number
genotype
SW040727
GII.3
SW040915
GII.2
SW050803
GII.4
sewage sludge 20 (1/5)
SS040727
GII.3
July 27, 2004
treated wastewater
20 (1/5)
TW050803
GII.4
Aug 3, 2005
river water
10 (2/20) RW050720St1 GII.4
July 20, 2005 July 20, 2005
RW050720St3 GII.4 gastroenteritis patients
FIGURE 1. Map of the research area. Circles indicate the sampling points for river water. The black rectangle is the location of the WWTP where the domestic wastewater, sewage sludge, and treated wastewater were collected. cation exchange resin and stirred lightly for 30 min at room temperature. Then, the suspension was centrifuged at 9000g for 30 min at 4 °C, and the supernatant was sampled. NoVs in the supernatant were concentrated by the polyethylene glycol precipitation. Concentrated samples were stored at -20 °C until further analysis. A stool sample was suspended in 9 times its weight of DDW and centrifuged at 9200g for 10 min. The supernatant was used for the extraction and amplification of the NoV GII gene. Extraction and Amplification of the NoV GII Gene. RNA was extracted from the concentrated samples with the QIAmp RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Complementary DNA synthesis and amplification of the NoV GII gene was performed as described previously (5). The sequence of the antisense primer used in RT-PCR was 5′-CCRCCNGCATRHCCRTTRTACAT-3′ (G2-SKR) (16). The sequences of the sense primers added in the first and second PCR were 5′CARGARBCNATGTTYAGRTGGATGAG-3′ (COG2F) and 5′CNTGGGAGGGCGATCGCAA-3′ (G2-SKF), respectively (16). The length of the products from the seminested PCR was 344 base pairs. Approximately 5 µL of the products from the seminested PCR were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. Cloning, Sequencing, and Phylogenetic Analysis of the NoV GII Gene. RT-PCR products were purified with the GFX PCR DNA and Gel Band Purification Kit (Amersham Bioscience, Piscataway, NJ). Purified products were cloned with pGEM-T Easy (Promega, Madison, WI) and E. coli DH5R (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. Three colonies were picked up from each RTPCR product, and the insertion sizes were checked by direct PCR amplifications with SP6 and T7 primers. Plasmids carrying the possible NoV gene were purified from E. coli cells with Miniprep kits (TaKaRa, Shiga, Japan), and the nucleotide sequence of the insertion was analyzed with the BigDye Terminator Cycle Sequencing protocol. The determined nucleotide sequences of the partial region of the capsid gene were aligned, and a phylogenetic tree with 1000 bootstrap replicates was generated by the neighbor-joining method with ClustalW (http://www.ddbj.nig.ac.jp/search/ clustalw-e.html). Norwalk virus, the prototype of NoV GI, was used as an outgroup. Bootstrap values of 950 or higher 7424
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collection date
HM040514-1
GII.4
HM040514-2
GII.4
HM040514-3
GII.4
HM040521
GII.4
HM040611
GII.2
HM050413
GII.4
HM050426
GII.4
HM050511
GII.4
HM050525-1
GII.4
HM050525-2
GII.4
HM050713
GII.4
July 27, 2004 Sept 15, 2004 Aug 3, 2005
May 14, 2004 May 14, 2004 May 14, 2004 May 21, 2004 June 11, 2004 April 13, 2005 April 26, 2005 May 11, 2005 May 25, 2005 May 25, 2005 July 13, 2005
a Sampling periods were between July and September in 2004 and between July and August in 2005.
were considered statistically significant for the grouping (17). The estimated number of nucleotide substitutions of the NoV GII.4 gene (241 nucleotides) were calculated with dnadist in the PHYLIP package (http://evolution.genetics.washington.edu/phylip.html). The 10 and 16 nucleotide sequences of the NoV GII.4 gene from environmental and stool samples in this study and in our previous study (5) were used for the calculation of the number of nucleotide substitutions. The 8 and 9 nucleotide sequences of the NoV GII.4 strains obtained in Japan and the other countries were acquired from public database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and used as reference nucleotide sequences.
Results and Discussion Detection of NoV GII Gene from Environmental Samples. The NoV GII gene in 400 mL of domestic wastewater and sewage sludge, 1 L of treated wastewater and river water, and stool samples of gastroenteritis patients was investigated. Table 1 shows the collection source, positive rate, collection date, genotype, and serial number of the NoV GII gene obtained in this study. The positive results for the NoV GII gene were not due to the contamination because the negative controls for RNA extraction and RT-PCR were negative (data not shown). Three out of five samples of domestic wastewater were positive for the NoV GII gene. Outbreaks of gastroenteritis
caused by NoVs were not reported in the area under investigation during the sampling periods, so the high positive rate of the NoV GII gene in domestic wastewater (60%) implies sporadic infectious diseases caused by NoV GII strains or asymptomatic patients have occurred in this area. However, positive rates of the NoV GII gene in sewage sludge and treated wastewater were 20% (one positive sample out of five samples), while our previous study showed that the positive rate of the NoV gene in treated wastewater at the same WWTP during the epidemic winter season was about 90% (5). Since exactly the same procedures were used in this study and the previous study, it is unlikely that the difference of the positive rates in the NoV detection is due to the presence of inhibitory substances for RT-PCR in environmental samples in this study. These results imply that the contamination of the water environment with NoVs has occurred throughout a year, although the frequency of NoV occurrence in this study period was considerably decreased compared to the epidemic winter season. The NoV GII gene was detected at sites 1 and 3 in July 2005. Site 1 is located on about 200 m upper stream of the discharge point of the treated wastewater; therefore, it is unlikely that the NoV GII gene obtained at site 1 originated from treated wastewater from the WWTP. A sewage system is not available in some districts around site 1, and each household in these regions has a water-purifier tank, in which the treated wastewater is directly discharged to surface water of the Takagi River. Not only the treated wastewater from the WWTP but also that from water-purifier tanks could be the contamination sources for NoVs in this area. The concomitance of multiple genotypes of NoVs in individual samples was not observed in this study, although several genotypes could be often obtained from water (9) and stool samples (11). We cloned the RT-PCR products and sequenced three individual clones, but the analysis of three clones might be insufficient to obtain multiple genotypes. However, it is possible to obtain a predominant genotype in each sample, which could be an important as an etiological agent. Phylogenetic Analysis of the NoV GII Capsid Gene. In this study, 18 nucleotide sequences of the NoV GII gene were collected in geographically close areas, which consist of 11 from stool samples of gastroenteritis patients, 3 from domestic sewage, 2 from river water, 1 from sewage sludge, and 1 from treated wastewater of the WWTP (Table 1). Figure 2 illustrates the result of phylogenetic analysis for the NoV gene. Fourteen out of eighteen nucleotide sequences of the NoV GII capsid gene (2 out of 5 sewage samples, 2 out of 2 river samples, and 10 out of 11 stool samples) belong to genotype 4 (GII.4). NoVs in GII.4 have caused the majority of NoV epidemics over the world (13, 18) and frequently emerged in the same area from December 2003 to January 2004 (5). These results mean that variants of NoV GII.4 have been prevalent and infected residents in this region throughout the year. Two nucleotide sequences of the NoV GII gene obtained from domestic wastewater and treated wastewater at the WWTP were estimated to be variants of Mexico virus (MX), the prototype of genotype 3 (NoV GII.3). Although several variants of NoV GII.3 have been observed in this area in two winter seasons, the detection frequency of NoV GII.3 was fairly lower than that of NoV GII.4 (5). It is reported that variants of MX recently caused a large outbreak in which 660 out of 1492 tourists who ate a lunch prepared by a restaurant in Nagasaki prefecture, Japan, were affected (19). Some researchers described that recombinants of MX and the other strains in NoV GII have naturally emerged (20, 21). Tsugawa et al. reported that a possible recombinant of MX and Hawaii virus in genotype 1 (GII.1) was found during the epidemic season from an outbreak in an infant home (January 2000),
FIGURE 2. Phylogenetic tree based on partial nucleotide sequences of the capsid gene (241 nucleotides) of NoVs. Black rectangles indicate the NoV GII gene obtained in the area under investigation. The distance was calculated by Kimura’s two-parameter method, and the tree was plotted by the neighbor-joining method. The phylogenetic tree was created by Njplot (http://pbil.univ-lyon1.fr/ software/njplot.html). Numbers at each branch indicate bootstrap values for the clusters supported by that branch. The Genebank accession numbers of the reference strains are as follows: MX, U22498; SMV, U70059; Hawai, U07611; Lordsdale, X86557; Amsterdam, AF195848; Southampton, L07418; Norwalk, M87661. and its clinical symptoms were more severe than those of previously reported sporadic or outbreak cases of NoV infections (20). The emergence of variants of MX in this study period implies that these viruses have been persistent and pose a risk for emerging new pathogenic recombinants of NoVs. Two nucleotide sequences of the NoV GII gene obtained from domestic sewage and gastroenteritis patients in this study period were categorized into genotype 2 (GII.2). NoV GII.2 includes the Snow Mountain agent (SMA) as the prototype of NoV GII (22). Strains in this genotype have not been predominant as etiological agents of gastroenteritis compared to those in GII.3 and GII.4. Kageyama et al. reported that diverse genotypes of NoVs have caused outbreaks of gastroenteritis in Saitama, Japan, but strains in NoV GII.2 were found only in several outbreaks that originated in semiclosed communities such as a school (11). NoV variants in GII.2 were also found in treated wastewater from the WWTP in January of 2004 in our previous study (5), which means this minor strain as an etiological agent of gastroenteritis has been persistent and infects residents in this area. VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Average of the estimated number of nucleotide substitutions in the NoV GII.4 gene (241 nucleotides) from environmental and stool samples in the area under investigation, the other districts in Japan, and the other countries. The error bars indicate the standard deviations. Accession numbers of nucleotide sequences of NoVs from Japan were as follows: AB186102 (Hu/ NV/OC03047/GII/2003/JP);DQ288291(Hu/GII/Nagano/2004/JP);DQ288292 (Hu/GII/Yamaguchi/2005/JP); DQ288293 (Hu/GII/Shizuoka/2005/JP); DQ288294 (Hu/GII/Hokkaido/2004/JP); DQ288295 (Hu/GII/Chiba/2004/ JP); DQ288296 (Hu/GII/Ehime/2005/JP); DQ288297 (Hu/GII/Hiroshima/ 2005/JP); DQ288298 (Hu/GII/Yamaguchi/2003/JP); DQ378864 (NV/GII/ Chiba1/2004/JP). Accession numbers of nucleotide sequences of NoVs from the other countries were as follows: AY485642 (NLV/ GII/Langen1061/2002/DE); AY502018 (Hu/NoV/CS/D1/2002/CAN); AY502023 (Hu/NoV/Farmington Hills/2002/USA); AY883096 (Hu/GII.4/ 2004/NL); DQ078801 (Hu/GII.4/Hunter 532D/04O/AU); DQ078829 (Hu/ GII.4/Sydney348/97O/AU); DQ157945 (Hu/GII/GpC/2004/Irl); DQ364459 (Hu/NoV/Lanzhou/35666/2002/China); DQ377175 (Hu/GII/VN981/2003/ VNM). Estimated Number of Nucleotide Substitutions in the Conservative Gene Region of NoV GII.4 Variants. Rates of spontaneous mutation of RNA viruses are much higher than those of DNA viruses and microbes (23), which brings about the high mutant frequency of RNA viruses. However, the N-terminal region of the NoV capsid protein is located on the interior of the viral particle (24), and the steric restriction of the N-terminal peptide inside of viral particle contributes to the conservation of its amino acid sequence. As a result, the sequence similarity of the N-terminal amino acid is relatively high compared to the other parts of whole sequence among NoV genogroups (25). Meanwhile, variants of NoV GII.4 have frequently infected humans compared to variants of the other NoV genotypes, in which base substitution mutations could be accumulated even in the conservative gene region. From this viewpoint, the number of nucleotide substitutions in the conservative gene region of NoV GII.4 were calculated to evaluate the genetic distance between NoV GII.4 variants. Figure 3 shows the average of the estimated number of nucleotide substitutions in the Nterminal gene region of the NoV GII.4 capsid protein. The 10 and 16 nucleotide sequences of the NoV GII.4 gene in environmental and stool samples obtained from the area under investigation between 2003 and 2005 were used for the calculation. On the other hand, eight and nine nucleotide sequences of NoV GII.4 strains originated from gastroenteritis patients in Japan and the other countries between 2003 and 2005 were used as reference nucleotide sequences. The averages of the number of nucleotide substitution in NoV GII gene from stool samples in the area under study, that in the other district in Japan and that in the other countries are 0.021, 0.020 and 0.020, respectively. On the other hand, the corresponding value for NoV GII.4 gene from environmental samples in the area under study was 0.031. Statistically significant differences between the numbers of nucleotide substitution in NoV GII.4 gene from environmental samples and those from stool samples were detected by Student’s 7426
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t-test at the rejection rate of 0.01. These results mean that genetically diverse strains of NoV GII.4 could be obtained from environmental samples compared to gastroenteritis patients. One of the reasons why NoVs in environmental samples differ from those in stool samples would be that environmental samples consist of NoVs derived from different persons that represent large populations, whereas stool samples represent single persons (8). In this study, we have shown that genetically diverse variants of NoVs, especially those in GII.4, were obtained from environmental samples. It could be possible to consecutively collect genetic information of NoV GII strains from water environment, while the continuous acquisition of NoV GII gene from gastroenteritis patients could be difficult because of the significant decrease in the cases of infectious diseases caused by NoVs in the non-epidemic season. Continual fixed-point observation of the genetic variations in the conservative gene region of NoV GII strains in water environment might provide important information on the rate of mutant frequency and the viral gene flow caused by the migration of human hosts, which could directly link to the elucidation of the year-round fate of NoVs.
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Received for review February 15, 2006. Revised manuscript received July 13, 2006. Accepted July 21, 2006. ES0603453
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