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Weekly variations in norovirus genogroup II genotypes in Japanese oysters
International Journal of Food Microbiology 284 (2018) 48–55
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Weekly variations in norovirus genogroup II genotypes in Japanese oysters a,b,⁎
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Jian Pu , Takayuki Miura , Shinobu Kazama , Yoshimitsu Konta , Nabila Dhyan Azraini , Erika Itoe, Hiroaki Itof, Tatsuo Omurac, Toru Watanabea a
Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan Faculty of Information Networking for Innovation and Design, Toyo University, Tokyo 115-0053, Japan New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi 980-8579, Japan d Faculty of Agricultural Technology, Gadjah Mada University, Yogyakarta 55281, Indonesia e Graduate School of Agricultural Science, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan f Center for Water Cycle, Marine Environment and Disaster Management, Kumamoto University, 2-39-1 Kurokami, Chuo-Ku, Kumamoto 860-8555, Japan b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Norovirus Massive parallel sequencing Molecular epidemiology Oyster Sewage
Increased levels of norovirus contamination in oysters were reportedly associated with a gastroenteritis epidemic occurring upstream of an oyster farming area. In this study, we monitored the norovirus concentration in oysters weekly between November 2014 and March 2015 and investigated the statistical relationship between norovirus genogroup II (GII) concentrations in oyster and sewage samples and the number of gastroenteritis cases in the area using cross-correlation analysis. A peak correlation coefficient (R = 0.76) at a time lag of +1 week was observed between the number of gastroenteritis cases and norovirus GII concentrations in oysters, indicating that oyster contamination is correlated with the number of gastroenteritis cases with a 1-week delay. Moreover, weekly variations in norovirus GII genotypes in oysters were evaluated using pyrosequencing. Only GII.3 was detected in November and December 2014, whereas GII.17 and GII.4 were present from January to March 2015. GII.17 Kawasaki 2014 strains were detected more frequently than GII.4 Sydney 2012 strains in oyster samples, as previously observed in stool and sewage samples collected during the same study period in Miyagi, Japan. Our observations indicate that there is a time lag between the circulation of norovirus genotypes in the human population and the detection of those genotypes in oysters.
1. Introduction Norovirus is a leading causative agent of acute gastroenteritis worldwide. Norovirus lineages can be classified into seven genogroups (GI-GVII), which can be further subdivided into at least 38 genotypes (Verhoef et al., 2015; Vinjé, 2015). Viruses belonging to GI, GII, and GIV are responsible for infection in humans, among which GII is the most prevalent genogroup worldwide (Medici et al., 2015; Zheng et al., 2006). A significant number of norovirus outbreaks have occurred since the mid-1990s, with the rapidly evolving GII.4 strain as the major cause. A novel GII variant, GII.17 Kawasaki 2014, emerged during the 2014–2015 norovirus season as a major cause of gastroenteritis outbreaks in Asia, and this variant replaced the previously dominant GII.4 Sydney 2012 variant (Chan et al., 2015; Matsushima et al., 2015). Among the 2133 cases reported in the Japan Infectious Agents Surveillance Report (IASR) from October 2014 to March 2015, GII.4, GII.3, and GII.17 were detected in 17.5%, 6.8%, and 4.7% of cases,
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respectively. A dramatic increase in the number of cases of GII.17 has been observed, from three in the 2013–2014 season to 100 in the 2014–2015 season in Japan (Matsushima et al., 2015). This lineage has been the predominant genotype since the beginning of the 2014–2015 winter season among both stool and sewage samples collected in Miyagi Prefecture, Japan (Kazama et al., 2017). Norovirus-associated outbreaks can involve both foodborne and person-to-person transmission. Noroviruses are increasingly becoming the most common cause of gastroenteritis infection associated with the consumption of shellfish (Schaeffer et al., 2013). However, the relative importance of transmission through shellfish as compared with that of person-to-person transmission is unknown. A systematic review of global outbreak surveillance data from 1999 to 2012 indicated that 14% of all norovirus outbreaks were caused by food (Verhoef et al., 2015). From September 2010 to August 2016, the ratio of foodborne norovirus outbreaks was even higher in Japan, which was 20%–34% (27% in average) based on data released by the National
Corresponding author at: Faculty of Information Networking for Innovation and Design, Toyo University, Tokyo, Japan. E-mail address: [email protected] (J. Pu). Present address: Department of Environmental Health, National Institute of Public Health, Saitama, Japan. 2 Present address: Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan. 1
https://doi.org/10.1016/j.ijfoodmicro.2018.06.027 Received 27 January 2018; Received in revised form 1 June 2018; Accepted 29 June 2018 Available online 05 July 2018 0168-1605/ © 2018 Elsevier B.V. All rights reserved.
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by vortexing for 10 s and centrifuged at 9100 ×g for 12 min. Approximately 50 μL of RNA was extracted from the supernatant using NucliSENS miniMAG reagents (BioMérieux, Marcy-l'Étoile, France) following the manufacturer's instructions. The RNA extracts were stored at −80 °C until further analysis.
Epidemiological Surveillance of Infectious Disease (NESID, 2017). Oysters are one of the most common shellfish eaten raw worldwide. According to the Family Income and Expenditure Survey Report, from 2013 to 2015, the average consumption of oysters per family in Japan was 493 g (Statistics Japan, 2016), which equals approximately 26 oysters assuming that the weight of a single shucked oyster is 19 g. Oysters can bioaccumulate human pathogenic microorganisms when grown in water impacted by sewage. Campos et al. (2017) predicted that an average of 130 sewage spills would lead to 500 copies of norovirus in 1 g of oyster based on a catchment population of 223,008, area of 72,953 ha, sewage spill volume of 1530 m3/day, and average distance between discharge and sampling point of 4 km. A review of norovirus contamination in shellfish in the UK revealed that among the 74,000 cases of norovirus associated with contaminated food annually, 16% (11,800 cases) were caused by oyster consumption (Hassard et al., 2017). Furthermore, oyster-related gastroenteritis is not always transmitted through locally or domestically harvested oysters. During January and February of 2016 in Denmark, 58 out of 67 persons were infected after consuming oysters harvested off the coast of La Rochelle, France (Rasmussen et al., 2016). Given the numerous reports of norovirus outbreaks in association with oyster consumption, oysters are considered one of the most important pathways for norovirus transmission (Flannery et al., 2012). In a previous study (Pu et al., 2016), we phylogenetically characterized norovirus isolates from two sewage and four oyster samples. The isolated strains of GII.17 from the sewage and oysters clustered with isolates derived from gastroenteritis cases in the GII.17 Kawasaki 2014 lineage, indicating a strong relationship among the prevalent strains. Thus, in this study, we aimed to improve our understanding of the temporal changes in norovirus genotypes circulating in the human population and those released into the environment in the 2014–2015 norovirus season. Miyagi prefecture, the second largest oyster production area in Japan, with an oyster yield as high as 20,000 tons per year in Matsushima Bay facing the Pacific Ocean, was selected as the target area for this study. We evaluated the correlation between the number of reported gastroenteritis cases and norovirus concentrations in sewage and oyster samples. Furthermore, we investigated variations in norovirus GII genotypes in weekly oyster samples using pyrosequencing.
2.2. Reverse transcription (RT) and quantitative real-time PCR (qPCR) RNA was reverse-transcribed into complementary DNA (cDNA) using an iScript Advanced cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) with a T100 thermal cycler (Bio-Rad) following the manufacturer's instructions. The RT reaction was conducted with the following protocol: 42 °C for 30 min for RT reaction, followed by 85 °C for 5 min for inactivation of reverse transcriptase. Oyster sample cDNA was stored at −80 °C for further analysis. For qPCR, the cDNAs were analyzed with a CFX96 Touch Real-Time PCR detection system (Bio-Rad) for the quantification of norovirus GII. qPCR was performed with 5 μL of cDNA and 15 μL of reaction mix containing Sso Advanced Universal Probes Supermix (Bio-Rad), together with primers and probes [COG2F, COG2R, and RING2AL-TP (5′FAMTGG GAG GGS GAT CGC RAT CT-TAMRA-3′)] as previously described (Kageyama et al., 2003; Kazama et al., 2016; Nakamura et al., 2010; Pu et al., 2016). PCR amplification was performed under the same conditions as those described by Kazama et al. (2016). A ten-fold dilution was prepared for each cDNA sample, and both undiluted and diluted samples were analyzed by qPCR. Tenfold serial dilutions (105–101 copies) of plasmid containing the target region were prepared for each standard curve (Kageyama et al., 2003). qPCR was performed in duplicate for both samples and standards. The DT of each oyster was spiked with murine norovirus (MNV, approximately 107 genome copies) during viral extraction, as a process control (Hata et al., 2011). MNV was also quantified by qPCR with the primers and probe described by Hata et al. (2011). The recovery rate for each sample was obtained by dividing the MNV amount measured by qPCR by the amount added to each oyster sample.
2.3. Quality controls for qPCR 2. Materials and methods For qPCR, samples with MNV recovery rates higher than 1% were considered validated for norovirus quantification (ISO 15216-1, 2017). Positive results were obtained from amplifications with fewer than 40 quantification cycles (Cq value < 40), in compliance with the MIQE guidelines (Bustin et al., 2009). The limit of detection (LOD) was approximately 1.1 log copies/g DT, based on the average oyster sample DT weight (calculated from three composite oyster samples each week for 18 weeks). Tubes used in all procedures, if not specifically mentioned, were sterilized polyethylene low-binding tubes.
2.1. Oyster sampling and viral RNA extraction Nine individual oyster samples were collected each Wednesday from November 5, 2014 to March 26, 2015 from a farming area in Miyagi Prefecture, Japan. The oyster samples were sent to the laboratory on ice within 24 h, and digestive tissue (DT) was excised immediately after arrival. Highest accumulations of noroviruses have been found in DT compared to other tissues in oysters, and thus it is often used for the detection of norovirus in oysters (Le Guyader et al., 2006; Tian et al., 2006; Wang et al., 2008). The DTs of the oyster samples were weighed (ranging from 2.1 to 4.9 g), and then each of the nine samples was placed in a 5-mL tube with two stainless steel beads (3.2 mm in diameter) and 1 mL enzyme solution containing 6.3 mg/mL α-amylase (A-3176 Type VI-B; Sigma, St. Louis, MO, USA), 0.25 mg/mL proteinase-K (#03115801001, PCR grade; Roche, Indianapolis, IN, USA), and 6.3 g/mL lipase (L1754 Type VII; Sigma). Each tube was processed on a Micro Smash-100 (TOMY, Tokyo, Japan) at 4200 rpm for 60 s, and samples were then incubated for 60 min at 37 °C, followed by 15 min at 60 °C. The mixture was then centrifuged at 9100 ×g for 12 min. The supernatants (approximately 4 mL) from three individual samples were mixed to form one composite sample. The three oyster composite samples produced each week were stored at −80 °C until subsequent analysis. For RNA extraction, 500 μL of 400 mM citrate buffer (pH 2.5) was added to 500 μL of each composite sample, and the samples were mixed
2.4. Nested PCR Considering the unquantifiable but possibly positive sample, one composite sample was selected from each of the eighteen weeks, with a Cq value below 45 according to qPCR. Sample cDNAs were used for nested PCR. The COG2F/G2SKR (CARGARBCNATGTTYAGRTGGATGAG/CCRCCNGCATRHCCRTTRTACAT) and G2SKF/G2SKR (CNTGGG AGGGCGATCGCAA/CCRCCNGCATRHCCRTTRTACAT) primer sets were used to amplify the capsid N/S-encoding domain (N/S) region using NebNext High Fidelity 2× PCR MasterMix (New England Biolabs, Ipswich, MA, USA), as described by Kazama et al. (2016). The nested PCR products were separated by agarose gel electrophoresis, and products with the expected length (344 bp) were excised from the gel and purified using a Qiagen Gel Extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. 49
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sewage samples with a lag of 0 to +3 weeks (R = 0.48–0.65; Fig. 2A); the maximum coefficient was found at a lag of 0 weeks (R = 0.65). As shown in Fig. 2B and C, changes in the concentration of norovirus GII in oyster samples followed those in sewage samples with a lag of −1 to +2 weeks (R = 0.55–0.80) and correlated with changes in the number of gastroenteritis cases with a lag of −1 to +2 weeks (R = 0.58–0.76). For the latter comparison, the peak correlation coefficient (R = 0.76) was observed at a lag of +1 week (Fig. 2C), indicating a 1-week delay between an increase in the number of gastroenteritis cases in the study area and an increase in oyster contamination.
2.5. Fusion PCR and pyrosequencing The purified nested PCR products were used for fusion PCR, and the fusion PCR products were purified using a Qiagen Gel Extraction Kit and evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). PCR products were then quantified, pyrosequenced, and subjected to bioinformatics analysis as described by Kazama et al. (2016). Phylogenetic analysis was carried out for GII genotypes in oyster samples, in comparison with those in sewage and stool samples, which were collected in the same study period and area. Nucleotide sequence data from oyster samples in the present study have been deposited in the DDBJ/EMBL/GenBank databases under the accession number DRA005610.
3.3. Analysis of norovirus GII genotypes distributed in oyster samples Among 18 weekly oyster composites, 17 passed the screening criteria for pyrosequencing, from which 119,282 reads were obtained. After de-noising, 0.02–8.3% of reads (1.4% on average) were removed during chimera analysis, resulting in approximately 7017 norovirus reads in each sample. An average of six operational taxonomic units (OTUs) per sample was obtained. All rarefaction curves reached a plateau, implying that we sampled the majority of the diversity of norovirus GII genotypes. A diversity of norovirus GII genotypes was found in the oysters in this study. Among the 17 oyster composites analyzed via pyrosequencing, five genotypes, GII.3, GII.4, GII.6, GII.13, and GII.17, were found in 100%, 29%, 5.9%, 5.9%, and 53% of the samples, respectively. The percentage of sequence reads belonging to each genotype or GII.4 subgenotype was calculated in each sample (Fig. 1). Genotypes GII.3 and GII.17 were the predominant genotypes in 71% and 24% of the 17 oyster composites, respectively. Notably, GII.17 was predominant in January and February 2015, whereas GII.3 was the leading genotype found in oysters at the other time points. Two GII.4 variants, Sydney 2012 and Den Haag 2006b, were detected in 29% (5/17) and 5.9% (1/ 17) of samples, respectively. The five samples harboring one or both of the two variants were collected in January and February 2015, and the Sydney 2012 variant was the predominant variant in four of these samples.
2.6. Cross-correlation analysis The norovirus GII concentrations of weekly oyster samples were compared with those in sewage samples and with weekly gastroenteritis cases reported during the same study period and in the same study area. Norovirus concentrations in sewage have been described previously by Kazama et al. (2017), with samples derived from influents collected during the same study period at the Matsushima wastewater treatment plant. Weekly gastroenteritis cases were reported by pediatric sentinel clinics (10 clinics) in the nearest administrative division, which included the study area (IDWR, 2017). Rotavirus-associated gastroenteritis cases were not included, as they have been reported separately since October 2013 in Japan. Thus, we assumed that most cases of gastroenteritis were caused by noroviruses (Kazama et al., 2017; NIID, 2017a). Time lags between the log-transformed norovirus GII concentrations in oyster and sewage samples and the number of gastroenteritis cases were investigated using cross-correlation analysis. A time-series cross-correlation coefficient of ± 7 weeks was calculated with SPSS 19 (SPSS, Inc., Chicago, IL, USA) with a significance level of 5%. The time lag ( ± 7 weeks) used in the cross-correlation analysis represents the time difference among the following events: 1) occurrence of gastroenteritis cases, 2) appearance of norovirus in sewage, and 3) contamination of oysters with norovirus.
3.4. Phylogenetic analysis of norovirus GII.3 and GII.17 in oyster samples 3. Results Phylogenetic analysis of the norovirus GII.3 genotypes found in oyster samples is shown in Fig. 3A. Sequential comparisons between GII.3 strains isolated from oyster and sewage (Kazama et al., 2017) samples suggested that the GII.3 strains collected from oyster samples could be divided into two subclusters. Most GII.3 strains isolated from oyster samples, as well as one isolated from a sewage sample, clustered closely with AB685707, which was obtained from a stool sample collected in a nursing home in Akita Prefecture, Japan in January 2011. Additional GII.3 strains were found in the oyster samples collected on January 14 and 22, 2015, and belonged to the same subcluster as a sewage sample collected on December 10, 2014. The sequence similarity was 100% between sequences derived from oysters and sewage. Phylogenetic analysis of GII.17 strains showed that the nucleotide sequences obtained from oyster samples were closely related to Kawasaki323 2014, forming a distinct subclade (Kawasaki308 2015) of the GII.17 Kawasaki 2014 lineage based on the N/S region sequences, as shown in Fig. 3B. Three strains (15FP2601SN, 15FP2605, and 15FP2607) obtained from three cases involved in an oyster-related outbreak in February 2015 in Miyagi Prefecture, as well as two strains isolated from two sewage samples collected in January and February 2015 (Pu et al., 2016), were used as references. Oyster samples were found to belong to the same subcluster (Kawasaki308 2015) as these references. Three GII.17 strains (15FP2601SN, 15FP2605, and 15FP2607) obtained from three stool samples in an oyster-related outbreak (in February 2015) in Miyagi Prefecture, and two strains isolated from two sewage samples collected in January and February 2015, have 98.0%–99.3% identity with GII.17 strains in oyster samples.
3.1. Quantitative detection of norovirus GII in oyster samples by RT-qPCR The quantification of norovirus GII genomes was considered valid in oyster composite samples with MNV recovery rates above 1%. Concentrations of norovirus GII genomes in oyster samples collected weekly from November 2014 to March 2015 ranged from 1.0 to 4.3 log10 copies/g DT, with a mean of 2.7 log10 copies/g DT (Fig. 1). Approximately 89% (48/54) of composite samples derived from 162 individual oysters were positive for norovirus GII. At least one positive sample was observed in each of the 18 weeks of the study. The peak concentration in the samples was observed in February 2015, which exhibited significantly higher concentrations compared with those of the other months [one-way analysis of variance (ANOVA) least significant difference [LSD], p < 0.02]. 3.2. Cross-correlation analysis between norovirus GII concentrations in oysters and sewage and number of gastroenteritis cases Using cross-correlation analysis, the norovirus GII concentrations in weekly oyster samples (n = 16, negative samples excluded) were compared with those in sewage samples described previously by Kazama et al. (2017) (n = 19) and with numbers of gastroenteritis cases reported weekly by 10 surveillance stations in the administrative division including the study area (IDWR, 2017) (Fig. 2). A significant correlation was found between the number of gastroenteritis cases in the study area and the log-transformed norovirus GII concentrations in 50
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Fig. 1. Weekly variations in norovirus GII concentrations and genotypes in oyster samples together with the number of gastroenteritis cases and norovirus GII concentrations in sewage. The dashed line indicates the limit of detection for oysters. The slashed lines in the genotype table indicate that pyrosequencing was not conducted for the sample. The asterisk (*) indicates the weeks in which no oyster samples were collected; (**) indicates that although the sample was positive by qPCR, the targeted peak was not observed in the Bioanalyzer step, and the sample was therefore not analyzed by pyrosequencing. “LOD” indicates limit of detection. Colors show the ranges of genotype distributions (%) in oyster samples: red, 100%; yellow, 10–100%; green, 1–10%; blue, < 1%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Discussion
who detected concentrations of norovirus that were 1 log-unit higher after cultivation of oysters at the discharge outfall of a wastewater treatment plant for 1 month. In an outbreak caused by oysters in France, the concentration of norovirus GII in oysters according to digital PCR was 43–950 copies per oyster (Polo et al., 2016), which is significantly lower than those observed in the present study (one-way ANOVA, LSD, p < 0.01). Our observations, together with evidence from previous studies, suggest that oysters serve as a crucial pathway of norovirus transmission and highlight the importance of monitoring norovirus levels in shellfish, as well as the sanitary quality of oyster farming areas. In addition, this study analyzed weekly variations in norovirus genotypes in oysters. In weekly samples from the same winter season, only two GII genotypes (GII.4 and GII.17) were observed among gastroenteritis cases, while much more genotypes were observed in sewage samples (GII.2, GII.3, GII.4, GII.6, GII.13, GII.14, and GII.17). This could be explained by the asymptomatic/mildly-symptomatic infection and the limitation of clinical surveillance. Sewage, as wastewater influent in this study, is a catchment receiving norovirus genotypes from human beings, thus reflecting norovirus circulation in human populations in a better way. Similar to the findings of Rajko-Nenow et al. (2012), we observed a diversity of norovirus GII genotypes in oysters (GII.3, GII.4, GII.6, GII.13, and GII.17), with GII.3 and GII.17 being the most frequently detected genotypes. Based on our analysis of the norovirus GII genotypes in oyster samples, combined with information on
In this study, we monitored the norovirus concentration in oysters weekly in Miyagi Prefecture between November 2014 and March 2015 and investigated the statistical relationship between norovirus GII concentrations in oyster and sewage samples and the number of gastroenteritis cases in the area using cross-correlation analysis. Based on our cross-correlation results, the maximum correlation coefficient (R = 0.65) between the number of gastroenteritis cases in the study area and norovirus GII concentrations in sewage samples was observed at a time lag of 0 weeks, consistent with a 3-year study (2013–2016) conducted by Kazama et al. (2017). Additionally, in the current study, the peak correlation coefficient (R = 0.76) between the number of gastroenteritis cases and norovirus concentrations in oyster was observed at a time lag of +1 week, indicating that oyster contamination levels followed the same trends as were observed for the number of gastroenteritis cases with a 1-week delay. Although norovirus contamination in oysters have been previously associated with a combined sewer overflow event (Flannery et al., 2013) or linked to gastroenteritis outbreaks (Le Mennec et al., 2017), in the present study, we obtained statistical evidence for a correlation between the variation in the number of gastroenteritis cases and variation in oyster norovirus GII concentrations through weekly monitoring during the 2014–2015 season. The concentrations of norovirus GII genomes detected in oysters in this study were lower than those described by Flannery et al. (2012), 51
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GII.4, GII.3 was found to be present in a much higher proportion than GII.4. This was consistent with the research findings of Maalouf et al. (2011), who found that the GII.4 strain tended to exhibit a lower bioaccumulation efficiency than GII.3 in oysters. Among five oyster samples harboring GII.4 collected in January and February 2015, all were found to contain the Sydney 2012 variant, while only one was found to contain the Den Haag 2006b variant. Notably, GII.17 was dominant during January and February 2015, whereas GII.3 was the leading genotype in oysters for the remaining time points in our study. Imamura et al. (2017) also identified GII.3 as the most abundant genotype detected in Japanese oysters collected from October to December 2015 but not from January to February 2016, by applying next-generation sequencing analysis. Considering the diversity of norovirus GII genotypes in oyster samples and the co-existence of multiple variants in the same oyster samples, ingestion of and infection with multiple genotypes may be possible and therefore increase the risk of infection in humans, since a mix of viral genotypes may result in the generation of new, recombinant viruses (Newell et al., 2010). For example, monthly observational data from the 2016–2017 winter season released by IASR, Japan showed a predominance of GII.2 among gastroenteritis cases from September 2016, with three types of recombinant strains (GII.Pe–GII.2, GII.P16–GII.2, and GII.P17–GII.2) emerging (NIID, 2017c; Thongprachum et al., 2017). Based on our pyrosequencing analysis, the GII.3 strains obtained from all oyster samples in which GII.3 was the most frequently detected genotype clustered in subcluster 1, close to strain AB685707, which was obtained from a stool sample collected in a nursing home in Akita Prefecture, Japan in January 2011. Interestingly, the GII.3 strains obtained from two oyster samples collected on January 14 and 22, 2015 belonged to subcluster 2, along with strains from three sewage samples (December 10, 2014; February 4, 2015; and March 4, 2015) collected during the 2014–2015 norovirus season. These findings demonstrate that oysters in the marine environment were contaminated with multiple strains derived from a single genotype. GII.17 strains have been reported globally from 1978 to 2013 (Hassard et al., 2017). According to the Infectious Agents Surveillance Report released by the Japan National Institute of Infectious Disease, GII.17 Kawasaki 2014 was detected for the first time in Japan in March 2014 in a gastroenteritis case in Kawasaki City that was the only case discovered during the 2013–2014 winter season in Japan (NIID, 2015). This strain was associated with the increased severity of clinical symptoms and exhibited high epidemic activity in September 2015 and August 2016. Although GII.4 remained the leading genotype in norovirus related outbreaks, accounting for 31.4% of reported cases, GII.17 was detected in 12.0% of cases (300/2504) in over 20 cities in Japan, becoming the second most common genotype after GII.4 (NIID, 2017b). Similar to observations in Japan, the novel GII.17 genotype (GII.17 Kawasaki 2014) has become the predominant genotype in other countries or regions in Asia. Indeed, among 23 outbreaks occurring in the 2014–2015 winter season in Jiangsu, China, 16 (69.6%) were related to GII.17 (Fu et al., 2015). During the same winter season, 82% of norovirus outbreaks in Guangdong, China were caused by the GII.17 variant (Lu et al., 2015). Moreover, Medici et al. (2015) identified GII.17 Kawasaki 2014 in Italy, and this novel strain was also detected in gastroenteritis cases in Europe, the United State, and Australia; however, this variant has not replaced the previously dominant GII.4 in these areas as it has in areas of Asia (de Graaf et al., 2015, 2016; Medici et al., 2015). In this study, GII.17 strains isolated from both gastroenteritis patients and sewage samples in the same study area clustered with Kawasaki308 2015 (Kazama et al., 2017), which originated in 2014 from a single haplotype during the initial emergence of in China and subsequently rapidly evolved into multiple sublineages globally (Chan et al., 2017). Prevalent norovirus strains were simultaneously discovered in oysters, sewage, and gastroenteritis cases during the 2014–2015 winter season, providing evidence that oysters were contaminated with GII.17 Kawasaki 2014 strains originating from sewage
Fig. 2. Distribution of cross-correlation coefficients between concentrations in sewage and the number of gastroenteritis cases (A), between norovirus GII concentrations in oysters and sewage (B), and between concentration in oysters and the number of gastroenteritis cases (C). While the events happened in the following order: 1) gastroenteritis cases were reported, 2) sewage became polluted by norovirus, and 3) oysters became polluted by norovirus, the time lag was defined as “+”. The two horizontal lines represent the 95% confidence interval for the correlation.
sewage samples taken weekly from related studies in Miyagi Prefecture during the 2014–2015 winter season (Kazama et al., 2017), the norovirus GII genotypes circulating in human populations were evidently detected in oysters, but the distribution of genotypes detected within a sample was different between sewage and gastroenteritis cases. The absence of the genotypes GII.2 and GII.14 in oysters, genotypes which showed low concentrations in sewage samples, may reflect their instability in the marine environment and possible selective accumulation resulting from specific binding ligands in oyster tissues. Several studies have demonstrated the selectivity of oysters in concentrating human pathogens and virus strains (Le Guyader et al., 2006, 2012; Maalouf et al., 2011). In our study, oyster samples containing both GII.3 and
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Fig. 3. Phylogenetic tree of the N/S regions of GII.3 (A) and GII.17 (B) strains obtained from oyster samples. Trees were built with the neighbor-joining method (K2+G), and bootstrapped with 1000 repetitions. (A) The GII.17 genotype strain (AY502009) was used as the outgroup. Four GII.3 strains isolated from four sewage samples collected on January 8, 2014; December 10, 2014; February 4, 2015; and March 4, 2015 were used for comparisons (Kazama et al., 2017). (B) The obtained sequences were designated with names starting from the sampling date (yymmdd) followed by “Oys” for oyster samples. “OTU1” and “OTU2” were added to the sample names when more than one variant was found in the same oyster sample. The reference sequences are designated with names following the order of the sampling year, strain name, location, and accession number.
circulation of norovirus genotypes in the human population and the detection of those genotypes in oysters. However, as pointed out by Randazzo et al. (2018), the RT-qPCR method used to quantify viral concentrations in this study may overestimate amounts of infectious viruses, as it detects viral RNA not only from infectious viral particles but also from inactivated ones. Thus, better quantification methods for infectious noroviruses in sewage and oyster samples are needed for a
and patients. Thus, our results suggest that additional treatment of wastewater is needed to ensure the safe production of oysters. In conclusion, a peak correlation between the number of gastroenteritis cases in the study area and the log-transformed norovirus GII concentration in oysters at a time lag of +1 week during the 2014–2015 norovirus season, as well as weekly variations in GII genotypes in oysters, indicated that there is a 1-week delay between the 53
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Fig. 3. (continued)
more rigorous analysis of oyster-associated infections, keeping in mind that the sanitary quality of oyster farming is of primary concern. Oysters represent a crucial pathway of norovirus transmission, and improving our understanding of the behaviors of viruses in oysters, as well as the virus/strain-selective mechanisms of oysters acting on viruses, may aid in the development of strategies for the prevention of infectious disease outbreaks.
J.H., Mans, J., Hung, T.N., Kwok, K., Cheung, K., Lee, N., Chan, P.K.S., 2017. Global spread of norovirus GII.17 Kawasaki 308, 2014–2016. Emerg. Infect. Dis. 23 (8), 1350–1354. de Graaf, M., van Beek, J., Vennema, H., Podkolzin, A.T., Hewitt, J., Bucardo, F., Templeton, K., Mans, J., Nordgren, J., Reuter, G., Lynch, M., Rasmussen, L.D., Iritani, N., Chan, M.C., Martella, V., Ambert-Balay, K., Vinjé, J., White, P.A., Koopmans, M.P., 2015. Emergence of a novel GII.17 norovirus - end of the GII.4 era? Euro Surveill. 20 (26), 21178. de Graaf, M., van Beek, J., Koopmans, M.P., 2016. Human norovirus transmission and evolution in a changing world. Nat. Rev. Microbiol. 14 (7), 421–433. Flannery, J., Keaveney, S., Rajko-Nenow, P., O'Flaherty, V., Doré, W., 2012. Concentration of norovirus during wastewater treatment and its impact on oyster contamination. Appl. Environ. Microbiol. 78 (9), 3400–3406. Flannery, J., Keaveney, S., Rajko-Nenow, P., O'Flaherty, V., Doré, W., 2013. Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters. Water Res. 47 (14), 5222–5231. Fu, J., Ai, J., Jin, M., Jiang, C., Zhang, J., Shi, C., Lin, Q., Yuan, Z., Qi, X., Bao, C., Tang, F., Zhu, Y., 2015. Emergence of a new GII.17 norovirus variant in patients with acute gastroenteritis in Jiangsu, China, September 2014 to March 2015. Euro Surveill. 20 (24), 21157. Hassard, F., Sharp, J.H., Taft, H., LeVay, L., Harris, J.P., McDonald, J.E., Tuson, K., Wilson, J., Jones, D.L., Malham, S.K., 2017. Critical review on the public health impact of norovirus contamination in shellfish and the environment: a UK perspective. Food Environ. Virol. 9 (2), 123–141. Hata, A., Katayama, H., Kitajima, M., Visvanathan, C., Nol, C., Furumai, H., 2011. Validation of internal controls for extraction and amplification of nucleic acids from enteric viruses in water samples. Appl. Environ. Microbiol. 77 (13), 4336–4343. IDWR, 2017. Infectious diseases weekly report of Miyagi Prefectural Government. http:// www.pref.miyagi.jp/site/hokans/surveypdf-shuho.html, Accessed date: 13 January 2017. Imamura, S., Kanezashi, H., Goshima, T., Haruna, M., Okada, T., Inagaki, N., Uema, M., Noda, M., Akimoto, K., 2017. Next-generation sequencing analysis of the diversity of human noroviruses in Japanese oysters. Foodborne Pathog. Dis. 14 (8), 465–471. ISO 15216-1:2017, 2017. Microbiology of the food chain-horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR- part 1: method for quantification. https://www.iso.org/standard/65681.html, Accessed date: 10 October 2017.
Acknowledgments This work was supported by a Core Research for Evolutional Science and Technology (CREST) program, “Innovation of water monitoring system with rapid, highly precise and exhaustive pathogen detection technologies”, of the Japan Science and Technology Agency (JST). References Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55 (4), 611–622. Campos, C.J.A., Kershaw, S., Morgan, O.C., Lees, D.N., 2017. Risk factors for norovirus contamination of shellfish water catchments in England and Wales. Int. J. Food Microbiol. 241, 318–324. Chan, M.C., Lee, N., Hung, T.N., Kwok, K., Cheung, K., Tin, E.K., Lai, R.W., Nelson, E.A., Leung, T.F., Chan, P.K., 2015. Rapid emergence and predominance of a broadly recognizing and fast-evolving norovirus GII.17 variant in late 2014. Nat. Commun. 6, 10061. Chan, M.C.W., Hu, Y.W., Chen, H.L., Podkolzin, A.T., Zaytseva, E.V., Komano, J., Sakon, N., Poovorawan, Y., Vongpunsawad, S., Thanusuwannasak, T., Hewitt, J., Croucher, D., Collins, N., Vinjé, J., Pang, X.L., Lee, B.E., de Graaf, M., van Beek, J., Vennema, H., Koopmans, M.P.G., Niendorf, S., Poljsak-Prijatelj, M., Steyer, A., White, P.A., Lun,
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Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Weekly variations in norovirus genogroup II genotypes in Japanese oysters a,b,⁎
c,1
c,2
c
T
d
Jian Pu , Takayuki Miura , Shinobu Kazama , Yoshimitsu Konta , Nabila Dhyan Azraini , Erika Itoe, Hiroaki Itof, Tatsuo Omurac, Toru Watanabea a
Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan Faculty of Information Networking for Innovation and Design, Toyo University, Tokyo 115-0053, Japan New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi 980-8579, Japan d Faculty of Agricultural Technology, Gadjah Mada University, Yogyakarta 55281, Indonesia e Graduate School of Agricultural Science, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan f Center for Water Cycle, Marine Environment and Disaster Management, Kumamoto University, 2-39-1 Kurokami, Chuo-Ku, Kumamoto 860-8555, Japan b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Norovirus Massive parallel sequencing Molecular epidemiology Oyster Sewage
Increased levels of norovirus contamination in oysters were reportedly associated with a gastroenteritis epidemic occurring upstream of an oyster farming area. In this study, we monitored the norovirus concentration in oysters weekly between November 2014 and March 2015 and investigated the statistical relationship between norovirus genogroup II (GII) concentrations in oyster and sewage samples and the number of gastroenteritis cases in the area using cross-correlation analysis. A peak correlation coefficient (R = 0.76) at a time lag of +1 week was observed between the number of gastroenteritis cases and norovirus GII concentrations in oysters, indicating that oyster contamination is correlated with the number of gastroenteritis cases with a 1-week delay. Moreover, weekly variations in norovirus GII genotypes in oysters were evaluated using pyrosequencing. Only GII.3 was detected in November and December 2014, whereas GII.17 and GII.4 were present from January to March 2015. GII.17 Kawasaki 2014 strains were detected more frequently than GII.4 Sydney 2012 strains in oyster samples, as previously observed in stool and sewage samples collected during the same study period in Miyagi, Japan. Our observations indicate that there is a time lag between the circulation of norovirus genotypes in the human population and the detection of those genotypes in oysters.
1. Introduction Norovirus is a leading causative agent of acute gastroenteritis worldwide. Norovirus lineages can be classified into seven genogroups (GI-GVII), which can be further subdivided into at least 38 genotypes (Verhoef et al., 2015; Vinjé, 2015). Viruses belonging to GI, GII, and GIV are responsible for infection in humans, among which GII is the most prevalent genogroup worldwide (Medici et al., 2015; Zheng et al., 2006). A significant number of norovirus outbreaks have occurred since the mid-1990s, with the rapidly evolving GII.4 strain as the major cause. A novel GII variant, GII.17 Kawasaki 2014, emerged during the 2014–2015 norovirus season as a major cause of gastroenteritis outbreaks in Asia, and this variant replaced the previously dominant GII.4 Sydney 2012 variant (Chan et al., 2015; Matsushima et al., 2015). Among the 2133 cases reported in the Japan Infectious Agents Surveillance Report (IASR) from October 2014 to March 2015, GII.4, GII.3, and GII.17 were detected in 17.5%, 6.8%, and 4.7% of cases,
⁎
respectively. A dramatic increase in the number of cases of GII.17 has been observed, from three in the 2013–2014 season to 100 in the 2014–2015 season in Japan (Matsushima et al., 2015). This lineage has been the predominant genotype since the beginning of the 2014–2015 winter season among both stool and sewage samples collected in Miyagi Prefecture, Japan (Kazama et al., 2017). Norovirus-associated outbreaks can involve both foodborne and person-to-person transmission. Noroviruses are increasingly becoming the most common cause of gastroenteritis infection associated with the consumption of shellfish (Schaeffer et al., 2013). However, the relative importance of transmission through shellfish as compared with that of person-to-person transmission is unknown. A systematic review of global outbreak surveillance data from 1999 to 2012 indicated that 14% of all norovirus outbreaks were caused by food (Verhoef et al., 2015). From September 2010 to August 2016, the ratio of foodborne norovirus outbreaks was even higher in Japan, which was 20%–34% (27% in average) based on data released by the National
Corresponding author at: Faculty of Information Networking for Innovation and Design, Toyo University, Tokyo, Japan. E-mail address: [email protected] (J. Pu). Present address: Department of Environmental Health, National Institute of Public Health, Saitama, Japan. 2 Present address: Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan. 1
https://doi.org/10.1016/j.ijfoodmicro.2018.06.027 Received 27 January 2018; Received in revised form 1 June 2018; Accepted 29 June 2018 Available online 05 July 2018 0168-1605/ © 2018 Elsevier B.V. All rights reserved.
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by vortexing for 10 s and centrifuged at 9100 ×g for 12 min. Approximately 50 μL of RNA was extracted from the supernatant using NucliSENS miniMAG reagents (BioMérieux, Marcy-l'Étoile, France) following the manufacturer's instructions. The RNA extracts were stored at −80 °C until further analysis.
Epidemiological Surveillance of Infectious Disease (NESID, 2017). Oysters are one of the most common shellfish eaten raw worldwide. According to the Family Income and Expenditure Survey Report, from 2013 to 2015, the average consumption of oysters per family in Japan was 493 g (Statistics Japan, 2016), which equals approximately 26 oysters assuming that the weight of a single shucked oyster is 19 g. Oysters can bioaccumulate human pathogenic microorganisms when grown in water impacted by sewage. Campos et al. (2017) predicted that an average of 130 sewage spills would lead to 500 copies of norovirus in 1 g of oyster based on a catchment population of 223,008, area of 72,953 ha, sewage spill volume of 1530 m3/day, and average distance between discharge and sampling point of 4 km. A review of norovirus contamination in shellfish in the UK revealed that among the 74,000 cases of norovirus associated with contaminated food annually, 16% (11,800 cases) were caused by oyster consumption (Hassard et al., 2017). Furthermore, oyster-related gastroenteritis is not always transmitted through locally or domestically harvested oysters. During January and February of 2016 in Denmark, 58 out of 67 persons were infected after consuming oysters harvested off the coast of La Rochelle, France (Rasmussen et al., 2016). Given the numerous reports of norovirus outbreaks in association with oyster consumption, oysters are considered one of the most important pathways for norovirus transmission (Flannery et al., 2012). In a previous study (Pu et al., 2016), we phylogenetically characterized norovirus isolates from two sewage and four oyster samples. The isolated strains of GII.17 from the sewage and oysters clustered with isolates derived from gastroenteritis cases in the GII.17 Kawasaki 2014 lineage, indicating a strong relationship among the prevalent strains. Thus, in this study, we aimed to improve our understanding of the temporal changes in norovirus genotypes circulating in the human population and those released into the environment in the 2014–2015 norovirus season. Miyagi prefecture, the second largest oyster production area in Japan, with an oyster yield as high as 20,000 tons per year in Matsushima Bay facing the Pacific Ocean, was selected as the target area for this study. We evaluated the correlation between the number of reported gastroenteritis cases and norovirus concentrations in sewage and oyster samples. Furthermore, we investigated variations in norovirus GII genotypes in weekly oyster samples using pyrosequencing.
2.2. Reverse transcription (RT) and quantitative real-time PCR (qPCR) RNA was reverse-transcribed into complementary DNA (cDNA) using an iScript Advanced cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) with a T100 thermal cycler (Bio-Rad) following the manufacturer's instructions. The RT reaction was conducted with the following protocol: 42 °C for 30 min for RT reaction, followed by 85 °C for 5 min for inactivation of reverse transcriptase. Oyster sample cDNA was stored at −80 °C for further analysis. For qPCR, the cDNAs were analyzed with a CFX96 Touch Real-Time PCR detection system (Bio-Rad) for the quantification of norovirus GII. qPCR was performed with 5 μL of cDNA and 15 μL of reaction mix containing Sso Advanced Universal Probes Supermix (Bio-Rad), together with primers and probes [COG2F, COG2R, and RING2AL-TP (5′FAMTGG GAG GGS GAT CGC RAT CT-TAMRA-3′)] as previously described (Kageyama et al., 2003; Kazama et al., 2016; Nakamura et al., 2010; Pu et al., 2016). PCR amplification was performed under the same conditions as those described by Kazama et al. (2016). A ten-fold dilution was prepared for each cDNA sample, and both undiluted and diluted samples were analyzed by qPCR. Tenfold serial dilutions (105–101 copies) of plasmid containing the target region were prepared for each standard curve (Kageyama et al., 2003). qPCR was performed in duplicate for both samples and standards. The DT of each oyster was spiked with murine norovirus (MNV, approximately 107 genome copies) during viral extraction, as a process control (Hata et al., 2011). MNV was also quantified by qPCR with the primers and probe described by Hata et al. (2011). The recovery rate for each sample was obtained by dividing the MNV amount measured by qPCR by the amount added to each oyster sample.
2.3. Quality controls for qPCR 2. Materials and methods For qPCR, samples with MNV recovery rates higher than 1% were considered validated for norovirus quantification (ISO 15216-1, 2017). Positive results were obtained from amplifications with fewer than 40 quantification cycles (Cq value < 40), in compliance with the MIQE guidelines (Bustin et al., 2009). The limit of detection (LOD) was approximately 1.1 log copies/g DT, based on the average oyster sample DT weight (calculated from three composite oyster samples each week for 18 weeks). Tubes used in all procedures, if not specifically mentioned, were sterilized polyethylene low-binding tubes.
2.1. Oyster sampling and viral RNA extraction Nine individual oyster samples were collected each Wednesday from November 5, 2014 to March 26, 2015 from a farming area in Miyagi Prefecture, Japan. The oyster samples were sent to the laboratory on ice within 24 h, and digestive tissue (DT) was excised immediately after arrival. Highest accumulations of noroviruses have been found in DT compared to other tissues in oysters, and thus it is often used for the detection of norovirus in oysters (Le Guyader et al., 2006; Tian et al., 2006; Wang et al., 2008). The DTs of the oyster samples were weighed (ranging from 2.1 to 4.9 g), and then each of the nine samples was placed in a 5-mL tube with two stainless steel beads (3.2 mm in diameter) and 1 mL enzyme solution containing 6.3 mg/mL α-amylase (A-3176 Type VI-B; Sigma, St. Louis, MO, USA), 0.25 mg/mL proteinase-K (#03115801001, PCR grade; Roche, Indianapolis, IN, USA), and 6.3 g/mL lipase (L1754 Type VII; Sigma). Each tube was processed on a Micro Smash-100 (TOMY, Tokyo, Japan) at 4200 rpm for 60 s, and samples were then incubated for 60 min at 37 °C, followed by 15 min at 60 °C. The mixture was then centrifuged at 9100 ×g for 12 min. The supernatants (approximately 4 mL) from three individual samples were mixed to form one composite sample. The three oyster composite samples produced each week were stored at −80 °C until subsequent analysis. For RNA extraction, 500 μL of 400 mM citrate buffer (pH 2.5) was added to 500 μL of each composite sample, and the samples were mixed
2.4. Nested PCR Considering the unquantifiable but possibly positive sample, one composite sample was selected from each of the eighteen weeks, with a Cq value below 45 according to qPCR. Sample cDNAs were used for nested PCR. The COG2F/G2SKR (CARGARBCNATGTTYAGRTGGATGAG/CCRCCNGCATRHCCRTTRTACAT) and G2SKF/G2SKR (CNTGGG AGGGCGATCGCAA/CCRCCNGCATRHCCRTTRTACAT) primer sets were used to amplify the capsid N/S-encoding domain (N/S) region using NebNext High Fidelity 2× PCR MasterMix (New England Biolabs, Ipswich, MA, USA), as described by Kazama et al. (2016). The nested PCR products were separated by agarose gel electrophoresis, and products with the expected length (344 bp) were excised from the gel and purified using a Qiagen Gel Extraction Kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. 49
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sewage samples with a lag of 0 to +3 weeks (R = 0.48–0.65; Fig. 2A); the maximum coefficient was found at a lag of 0 weeks (R = 0.65). As shown in Fig. 2B and C, changes in the concentration of norovirus GII in oyster samples followed those in sewage samples with a lag of −1 to +2 weeks (R = 0.55–0.80) and correlated with changes in the number of gastroenteritis cases with a lag of −1 to +2 weeks (R = 0.58–0.76). For the latter comparison, the peak correlation coefficient (R = 0.76) was observed at a lag of +1 week (Fig. 2C), indicating a 1-week delay between an increase in the number of gastroenteritis cases in the study area and an increase in oyster contamination.
2.5. Fusion PCR and pyrosequencing The purified nested PCR products were used for fusion PCR, and the fusion PCR products were purified using a Qiagen Gel Extraction Kit and evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). PCR products were then quantified, pyrosequenced, and subjected to bioinformatics analysis as described by Kazama et al. (2016). Phylogenetic analysis was carried out for GII genotypes in oyster samples, in comparison with those in sewage and stool samples, which were collected in the same study period and area. Nucleotide sequence data from oyster samples in the present study have been deposited in the DDBJ/EMBL/GenBank databases under the accession number DRA005610.
3.3. Analysis of norovirus GII genotypes distributed in oyster samples Among 18 weekly oyster composites, 17 passed the screening criteria for pyrosequencing, from which 119,282 reads were obtained. After de-noising, 0.02–8.3% of reads (1.4% on average) were removed during chimera analysis, resulting in approximately 7017 norovirus reads in each sample. An average of six operational taxonomic units (OTUs) per sample was obtained. All rarefaction curves reached a plateau, implying that we sampled the majority of the diversity of norovirus GII genotypes. A diversity of norovirus GII genotypes was found in the oysters in this study. Among the 17 oyster composites analyzed via pyrosequencing, five genotypes, GII.3, GII.4, GII.6, GII.13, and GII.17, were found in 100%, 29%, 5.9%, 5.9%, and 53% of the samples, respectively. The percentage of sequence reads belonging to each genotype or GII.4 subgenotype was calculated in each sample (Fig. 1). Genotypes GII.3 and GII.17 were the predominant genotypes in 71% and 24% of the 17 oyster composites, respectively. Notably, GII.17 was predominant in January and February 2015, whereas GII.3 was the leading genotype found in oysters at the other time points. Two GII.4 variants, Sydney 2012 and Den Haag 2006b, were detected in 29% (5/17) and 5.9% (1/ 17) of samples, respectively. The five samples harboring one or both of the two variants were collected in January and February 2015, and the Sydney 2012 variant was the predominant variant in four of these samples.
2.6. Cross-correlation analysis The norovirus GII concentrations of weekly oyster samples were compared with those in sewage samples and with weekly gastroenteritis cases reported during the same study period and in the same study area. Norovirus concentrations in sewage have been described previously by Kazama et al. (2017), with samples derived from influents collected during the same study period at the Matsushima wastewater treatment plant. Weekly gastroenteritis cases were reported by pediatric sentinel clinics (10 clinics) in the nearest administrative division, which included the study area (IDWR, 2017). Rotavirus-associated gastroenteritis cases were not included, as they have been reported separately since October 2013 in Japan. Thus, we assumed that most cases of gastroenteritis were caused by noroviruses (Kazama et al., 2017; NIID, 2017a). Time lags between the log-transformed norovirus GII concentrations in oyster and sewage samples and the number of gastroenteritis cases were investigated using cross-correlation analysis. A time-series cross-correlation coefficient of ± 7 weeks was calculated with SPSS 19 (SPSS, Inc., Chicago, IL, USA) with a significance level of 5%. The time lag ( ± 7 weeks) used in the cross-correlation analysis represents the time difference among the following events: 1) occurrence of gastroenteritis cases, 2) appearance of norovirus in sewage, and 3) contamination of oysters with norovirus.
3.4. Phylogenetic analysis of norovirus GII.3 and GII.17 in oyster samples 3. Results Phylogenetic analysis of the norovirus GII.3 genotypes found in oyster samples is shown in Fig. 3A. Sequential comparisons between GII.3 strains isolated from oyster and sewage (Kazama et al., 2017) samples suggested that the GII.3 strains collected from oyster samples could be divided into two subclusters. Most GII.3 strains isolated from oyster samples, as well as one isolated from a sewage sample, clustered closely with AB685707, which was obtained from a stool sample collected in a nursing home in Akita Prefecture, Japan in January 2011. Additional GII.3 strains were found in the oyster samples collected on January 14 and 22, 2015, and belonged to the same subcluster as a sewage sample collected on December 10, 2014. The sequence similarity was 100% between sequences derived from oysters and sewage. Phylogenetic analysis of GII.17 strains showed that the nucleotide sequences obtained from oyster samples were closely related to Kawasaki323 2014, forming a distinct subclade (Kawasaki308 2015) of the GII.17 Kawasaki 2014 lineage based on the N/S region sequences, as shown in Fig. 3B. Three strains (15FP2601SN, 15FP2605, and 15FP2607) obtained from three cases involved in an oyster-related outbreak in February 2015 in Miyagi Prefecture, as well as two strains isolated from two sewage samples collected in January and February 2015 (Pu et al., 2016), were used as references. Oyster samples were found to belong to the same subcluster (Kawasaki308 2015) as these references. Three GII.17 strains (15FP2601SN, 15FP2605, and 15FP2607) obtained from three stool samples in an oyster-related outbreak (in February 2015) in Miyagi Prefecture, and two strains isolated from two sewage samples collected in January and February 2015, have 98.0%–99.3% identity with GII.17 strains in oyster samples.
3.1. Quantitative detection of norovirus GII in oyster samples by RT-qPCR The quantification of norovirus GII genomes was considered valid in oyster composite samples with MNV recovery rates above 1%. Concentrations of norovirus GII genomes in oyster samples collected weekly from November 2014 to March 2015 ranged from 1.0 to 4.3 log10 copies/g DT, with a mean of 2.7 log10 copies/g DT (Fig. 1). Approximately 89% (48/54) of composite samples derived from 162 individual oysters were positive for norovirus GII. At least one positive sample was observed in each of the 18 weeks of the study. The peak concentration in the samples was observed in February 2015, which exhibited significantly higher concentrations compared with those of the other months [one-way analysis of variance (ANOVA) least significant difference [LSD], p < 0.02]. 3.2. Cross-correlation analysis between norovirus GII concentrations in oysters and sewage and number of gastroenteritis cases Using cross-correlation analysis, the norovirus GII concentrations in weekly oyster samples (n = 16, negative samples excluded) were compared with those in sewage samples described previously by Kazama et al. (2017) (n = 19) and with numbers of gastroenteritis cases reported weekly by 10 surveillance stations in the administrative division including the study area (IDWR, 2017) (Fig. 2). A significant correlation was found between the number of gastroenteritis cases in the study area and the log-transformed norovirus GII concentrations in 50
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Fig. 1. Weekly variations in norovirus GII concentrations and genotypes in oyster samples together with the number of gastroenteritis cases and norovirus GII concentrations in sewage. The dashed line indicates the limit of detection for oysters. The slashed lines in the genotype table indicate that pyrosequencing was not conducted for the sample. The asterisk (*) indicates the weeks in which no oyster samples were collected; (**) indicates that although the sample was positive by qPCR, the targeted peak was not observed in the Bioanalyzer step, and the sample was therefore not analyzed by pyrosequencing. “LOD” indicates limit of detection. Colors show the ranges of genotype distributions (%) in oyster samples: red, 100%; yellow, 10–100%; green, 1–10%; blue, < 1%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Discussion
who detected concentrations of norovirus that were 1 log-unit higher after cultivation of oysters at the discharge outfall of a wastewater treatment plant for 1 month. In an outbreak caused by oysters in France, the concentration of norovirus GII in oysters according to digital PCR was 43–950 copies per oyster (Polo et al., 2016), which is significantly lower than those observed in the present study (one-way ANOVA, LSD, p < 0.01). Our observations, together with evidence from previous studies, suggest that oysters serve as a crucial pathway of norovirus transmission and highlight the importance of monitoring norovirus levels in shellfish, as well as the sanitary quality of oyster farming areas. In addition, this study analyzed weekly variations in norovirus genotypes in oysters. In weekly samples from the same winter season, only two GII genotypes (GII.4 and GII.17) were observed among gastroenteritis cases, while much more genotypes were observed in sewage samples (GII.2, GII.3, GII.4, GII.6, GII.13, GII.14, and GII.17). This could be explained by the asymptomatic/mildly-symptomatic infection and the limitation of clinical surveillance. Sewage, as wastewater influent in this study, is a catchment receiving norovirus genotypes from human beings, thus reflecting norovirus circulation in human populations in a better way. Similar to the findings of Rajko-Nenow et al. (2012), we observed a diversity of norovirus GII genotypes in oysters (GII.3, GII.4, GII.6, GII.13, and GII.17), with GII.3 and GII.17 being the most frequently detected genotypes. Based on our analysis of the norovirus GII genotypes in oyster samples, combined with information on
In this study, we monitored the norovirus concentration in oysters weekly in Miyagi Prefecture between November 2014 and March 2015 and investigated the statistical relationship between norovirus GII concentrations in oyster and sewage samples and the number of gastroenteritis cases in the area using cross-correlation analysis. Based on our cross-correlation results, the maximum correlation coefficient (R = 0.65) between the number of gastroenteritis cases in the study area and norovirus GII concentrations in sewage samples was observed at a time lag of 0 weeks, consistent with a 3-year study (2013–2016) conducted by Kazama et al. (2017). Additionally, in the current study, the peak correlation coefficient (R = 0.76) between the number of gastroenteritis cases and norovirus concentrations in oyster was observed at a time lag of +1 week, indicating that oyster contamination levels followed the same trends as were observed for the number of gastroenteritis cases with a 1-week delay. Although norovirus contamination in oysters have been previously associated with a combined sewer overflow event (Flannery et al., 2013) or linked to gastroenteritis outbreaks (Le Mennec et al., 2017), in the present study, we obtained statistical evidence for a correlation between the variation in the number of gastroenteritis cases and variation in oyster norovirus GII concentrations through weekly monitoring during the 2014–2015 season. The concentrations of norovirus GII genomes detected in oysters in this study were lower than those described by Flannery et al. (2012), 51
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GII.4, GII.3 was found to be present in a much higher proportion than GII.4. This was consistent with the research findings of Maalouf et al. (2011), who found that the GII.4 strain tended to exhibit a lower bioaccumulation efficiency than GII.3 in oysters. Among five oyster samples harboring GII.4 collected in January and February 2015, all were found to contain the Sydney 2012 variant, while only one was found to contain the Den Haag 2006b variant. Notably, GII.17 was dominant during January and February 2015, whereas GII.3 was the leading genotype in oysters for the remaining time points in our study. Imamura et al. (2017) also identified GII.3 as the most abundant genotype detected in Japanese oysters collected from October to December 2015 but not from January to February 2016, by applying next-generation sequencing analysis. Considering the diversity of norovirus GII genotypes in oyster samples and the co-existence of multiple variants in the same oyster samples, ingestion of and infection with multiple genotypes may be possible and therefore increase the risk of infection in humans, since a mix of viral genotypes may result in the generation of new, recombinant viruses (Newell et al., 2010). For example, monthly observational data from the 2016–2017 winter season released by IASR, Japan showed a predominance of GII.2 among gastroenteritis cases from September 2016, with three types of recombinant strains (GII.Pe–GII.2, GII.P16–GII.2, and GII.P17–GII.2) emerging (NIID, 2017c; Thongprachum et al., 2017). Based on our pyrosequencing analysis, the GII.3 strains obtained from all oyster samples in which GII.3 was the most frequently detected genotype clustered in subcluster 1, close to strain AB685707, which was obtained from a stool sample collected in a nursing home in Akita Prefecture, Japan in January 2011. Interestingly, the GII.3 strains obtained from two oyster samples collected on January 14 and 22, 2015 belonged to subcluster 2, along with strains from three sewage samples (December 10, 2014; February 4, 2015; and March 4, 2015) collected during the 2014–2015 norovirus season. These findings demonstrate that oysters in the marine environment were contaminated with multiple strains derived from a single genotype. GII.17 strains have been reported globally from 1978 to 2013 (Hassard et al., 2017). According to the Infectious Agents Surveillance Report released by the Japan National Institute of Infectious Disease, GII.17 Kawasaki 2014 was detected for the first time in Japan in March 2014 in a gastroenteritis case in Kawasaki City that was the only case discovered during the 2013–2014 winter season in Japan (NIID, 2015). This strain was associated with the increased severity of clinical symptoms and exhibited high epidemic activity in September 2015 and August 2016. Although GII.4 remained the leading genotype in norovirus related outbreaks, accounting for 31.4% of reported cases, GII.17 was detected in 12.0% of cases (300/2504) in over 20 cities in Japan, becoming the second most common genotype after GII.4 (NIID, 2017b). Similar to observations in Japan, the novel GII.17 genotype (GII.17 Kawasaki 2014) has become the predominant genotype in other countries or regions in Asia. Indeed, among 23 outbreaks occurring in the 2014–2015 winter season in Jiangsu, China, 16 (69.6%) were related to GII.17 (Fu et al., 2015). During the same winter season, 82% of norovirus outbreaks in Guangdong, China were caused by the GII.17 variant (Lu et al., 2015). Moreover, Medici et al. (2015) identified GII.17 Kawasaki 2014 in Italy, and this novel strain was also detected in gastroenteritis cases in Europe, the United State, and Australia; however, this variant has not replaced the previously dominant GII.4 in these areas as it has in areas of Asia (de Graaf et al., 2015, 2016; Medici et al., 2015). In this study, GII.17 strains isolated from both gastroenteritis patients and sewage samples in the same study area clustered with Kawasaki308 2015 (Kazama et al., 2017), which originated in 2014 from a single haplotype during the initial emergence of in China and subsequently rapidly evolved into multiple sublineages globally (Chan et al., 2017). Prevalent norovirus strains were simultaneously discovered in oysters, sewage, and gastroenteritis cases during the 2014–2015 winter season, providing evidence that oysters were contaminated with GII.17 Kawasaki 2014 strains originating from sewage
Fig. 2. Distribution of cross-correlation coefficients between concentrations in sewage and the number of gastroenteritis cases (A), between norovirus GII concentrations in oysters and sewage (B), and between concentration in oysters and the number of gastroenteritis cases (C). While the events happened in the following order: 1) gastroenteritis cases were reported, 2) sewage became polluted by norovirus, and 3) oysters became polluted by norovirus, the time lag was defined as “+”. The two horizontal lines represent the 95% confidence interval for the correlation.
sewage samples taken weekly from related studies in Miyagi Prefecture during the 2014–2015 winter season (Kazama et al., 2017), the norovirus GII genotypes circulating in human populations were evidently detected in oysters, but the distribution of genotypes detected within a sample was different between sewage and gastroenteritis cases. The absence of the genotypes GII.2 and GII.14 in oysters, genotypes which showed low concentrations in sewage samples, may reflect their instability in the marine environment and possible selective accumulation resulting from specific binding ligands in oyster tissues. Several studies have demonstrated the selectivity of oysters in concentrating human pathogens and virus strains (Le Guyader et al., 2006, 2012; Maalouf et al., 2011). In our study, oyster samples containing both GII.3 and
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Fig. 3. Phylogenetic tree of the N/S regions of GII.3 (A) and GII.17 (B) strains obtained from oyster samples. Trees were built with the neighbor-joining method (K2+G), and bootstrapped with 1000 repetitions. (A) The GII.17 genotype strain (AY502009) was used as the outgroup. Four GII.3 strains isolated from four sewage samples collected on January 8, 2014; December 10, 2014; February 4, 2015; and March 4, 2015 were used for comparisons (Kazama et al., 2017). (B) The obtained sequences were designated with names starting from the sampling date (yymmdd) followed by “Oys” for oyster samples. “OTU1” and “OTU2” were added to the sample names when more than one variant was found in the same oyster sample. The reference sequences are designated with names following the order of the sampling year, strain name, location, and accession number.
circulation of norovirus genotypes in the human population and the detection of those genotypes in oysters. However, as pointed out by Randazzo et al. (2018), the RT-qPCR method used to quantify viral concentrations in this study may overestimate amounts of infectious viruses, as it detects viral RNA not only from infectious viral particles but also from inactivated ones. Thus, better quantification methods for infectious noroviruses in sewage and oyster samples are needed for a
and patients. Thus, our results suggest that additional treatment of wastewater is needed to ensure the safe production of oysters. In conclusion, a peak correlation between the number of gastroenteritis cases in the study area and the log-transformed norovirus GII concentration in oysters at a time lag of +1 week during the 2014–2015 norovirus season, as well as weekly variations in GII genotypes in oysters, indicated that there is a 1-week delay between the 53
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Fig. 3. (continued)
more rigorous analysis of oyster-associated infections, keeping in mind that the sanitary quality of oyster farming is of primary concern. Oysters represent a crucial pathway of norovirus transmission, and improving our understanding of the behaviors of viruses in oysters, as well as the virus/strain-selective mechanisms of oysters acting on viruses, may aid in the development of strategies for the prevention of infectious disease outbreaks.
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