Application of Next-Generation Sequencing to Evaluate the Profile of Noroviruses in Pre- and Post-Depurated Oysters

Abstract

The development of procedures for the efficient removal or inactivation of noroviruses from contaminated oysters is of great interest in oyster production. However, there is a critical limitation for evaluating the depuration efficacy of presently available procedures, as no suitable cell culture system currently exists to cultivate noroviruses. Thus, we applied a next-generation sequencing (NGS) technique to characterize norovirus genotypes in pre- and post-depurated oysters. As a result, we revealed the diversity of noroviruses in pre- and post-depurated oysters. Although the applied depuration procedure could reduce the number of bacterial agents to the level recommended by the Japanese Ministry of Health, Labour and Welfare, no significant changes were observed in the detection rate and the proportion of norovirus group (G) I and GII genotypes. To our knowledge, this is the first report to evaluate the profile of noroviruses in pre- and post-depurated oysters, specifically with respect to norovirus removal, using NGS; the findings imply that the removal of noroviruses from oysters through depuration is not presently sufficient. Further studies are needed to develop a more suitable depuration procedure for removing and/or inactivating noroviruses from contaminated oysters.

Introduction

Noroviruses have been major causative agents of acute gastroenteritis and have significant impacts on human health (Kingsley et al., 2002; EFSA, 2011). Human activities, involving sewage disposal, contribute greatly to the contamination of the environment with noroviruses (Maalouf et al., 2010). Through heavy rains or melting snow, noroviruses are transported to rivers and seas (Gentry et al., 2009; Wyn-Jones et al., 2011), which may result in the contamination of aquatic environments. As filter feeders, shellfish can readily accumulate different contaminants such as bacteria and viruses from surrounding water (Oliveira et al., 2011). Consequently, shellfish are known to be potential vectors for pathogens (Montazeri et al., 2015). In addition, some norovirus strains bind to shellfish tissues using carbohydrate structures similar to their human ligands (Maalouf et al., 2011), leading to the hypothesis of ineffectiveness of depuration procedures.

Developing procedures for efficient removal or inactivation of pathogenic viruses from contaminated oysters are of interest to improve food safety (Lees, 2000; Calci et al., 2005). Viral surrogates have been used to evaluate the efficacy of norovirus removal or inactivation in oysters, as there are no suitable cell culture systems to cultivate these viruses (Wobus et al., 2004). Feline calicivirus (FCV) is one of the most frequently used surrogates (Wollants et al., 2004; Kampf et al., 2005). However, Ueki et al. (2007) demonstrated that FCV accumulation in the oyster was promptly eliminated from the digestive diverticulum of artificially contaminated oysters, whereas noroviruses persisted in the oyster body even 10-d after depuration. Thus, evaluating the efficacy of norovirus removal through depuration is highly dependent on the performance of the virus surrogate. Nowadays, murine norovirus (MNV) and Tulane virus (TV) are shown to be optimal surrogates for their similarities to norovirus and for their relative ease of use in laboratory (Kniel et al., 2014). MNV is a true norovirus and showed greater genetic similarity to human norovirus (Farkas et al., 2008; Donaldson et al., 2010). On the contrary, TV has similar histo-blood binding group antigen binding properties to human noroviruses (Tian et al., 2006; Hirneisen and Kniel, 2013); however, selection of a better viral surrogate for noroviruses is ongoing, since these surrogate viruses still have some limitations.

Meanwhile, several studies have used next-generation sequencing (NGS) in an attempt to describe the full range of microbes present in foods, as well as to understand temporal microflora changes during various steps of procedures (O'Sullivan, 2000; Lusk et al., 2012). Among them, we first reported application of NGS to investigation of norovirus diversity in shellfish collected from coastal sites in Japan from 2013 to 2014 (Imamura et al., 2016). In this study, we utilize the latest technology to reveal diversity of norovirus genotypes present in pre- and post-depurated oysters, since we hypothesize that the elimination of noroviruses in oysters by depuration could be represented by alterations of genotype profiles in the sample. Accordingly, the current study aimed to investigate the efficacy of elimination of noroviruses from oysters through depuration, determined using NGS, and to suggest ways to improve sanitary level for noroviruses in oysters.

Materials and Methods

Oysters

Oysters (Crassostrea gigas) were supplied by a fish producer once a week from site A, which is one of the main production areas in East Japan during January to March, 2015. Oysters, which were 1-year-old at the time of the study, were collected from offshore area of oyster treatment plant of the fish producer.

Depuration system and water

The fish producer has a depuration system nearby their oyster treatment plant. Seawater was mechanically pumped into the tank for sterilization, wherein the seawater was treated with a 240 W UV sterilizer (Aqua Ultraviolet) for 1 h. The sterilization tank produced 74,000 L of sterilized seawater per hour. Next, the sterilized water was streamed into the tank for depuration, wherein oysters were suspended in a middle position from the bottom of the tank in a monolayer on a mesh (2.5 cm) container, at ambient temperature for more than 18 h. This system was regulated such that at least 720 L/h of sterilized sweater was pumped in for every 1000 oysters.

A 100 mL water sample was collected from the sea nearby the depuration system, while 100 mL of sterilized water was collected directly from the depuration system. Both water samples were examined for coliform bacteria.

Sample preparation for bacterial examinations

Twenty oysters were shucked and assembled to prepare the sample for bacterial examinations. These oysters were cut into small pieces and homogenized in equal volumes of phosphate-buffered saline (PBS) using stomachere.

Aerobic plate count

The sample was diluted up to 1000-fold with PBS for the aerobic plate count (APC). One milliliter of each dilution was used for pour-plate culture (standard method agar Nissui [https://cosmokai.com/youran/05618.pdf]; 1 L contains 5.0 g of peptone, 2.5 g of yeast, 1.0 g of glucose, and 15.0 g of agar; Nissui, Tokyo, Japan) and incubated at 35°C for 48 h. The number of colonies was determined by visual observation.

Most probable number of the fecal coliforms and Vibrio parahaemolyticus

Fecal coliform load was determined according to the 5-tube most probable number (MPN) method based on the American Public Health Association Standard Method (1998). Briefly, the samples were inoculated into broth (EC broth Nissui [https://cosmokai.com/youran/05618.pdf]; 1 L contains 20.0 g of peptone, 5.0 g of lactose, 1.5 g of bile salts, 4.0 g of dibasic potassium phosphate, 1.5 g of potassium dihydrogen phosphate, and 5.0 g of sodium chloride; Nissui) and were incubated in a water bath at 44.5°C for 24 h. Visual observation was conducted to confirm gas production in Durham tube.

Vibrio parahaemolyticus load was determined according to the 3-tube MPN method described in the Standards and criteria for food and food additives, etc., under the Food Sanitation Law (1947). Briefly, the samples were precultured in alkaline peptone broth (Nissui) at 37°C overnight. To identify candidates of V. parahaemolyticus, enrichments were plated on thiosulfate citrate bile salts sucrose agar (Nissui) and incubated at 37°C overnight. Following identification, MPN procedure was conducted for the enumeration.

MPN of total coliform bacteria

Total coliform bacteria load in seawater was determined according to the 3-tube MPN method described in the Standards and criteria for food and food additives, etc., under the Food Sanitation Law. Briefly, seawater was incubated in lactose broth (Nissui) at 48°C overnight. The seawater that produced gas in LB was determined as candidate positive samples of coliform bacteria. These culture media were inoculated into brilliant green-lactose-bile (BGLB) broth (DAIGO) and incubated at 35°C for 48 h. Seawater that produced gas in BGLB broth was determined to be a positive sample of coliform bacteria. Following the determination, the MPN procedure was conducted for enumeration.

Sample preparation of viral suspensions for genetic analyses

The oysters were sent to the laboratory and refrigerated (4°C) within 24 h of harvest or depuration. For the preparation of viral suspensions, oysters were shucked and the digestive diverticula were removed by dissection on the day of arrival to the laboratory. Three dissected digestive diverticula were combined as one sample, so that the combined mass was greater than 2.0 ± 0.2 g as described elsewhere (ISO/TS 15216-1, 2013). The combined sample was homogenized in a volume of PBS solution (without magnesium and calcium) nine times the sample weight (Le Guyader et al., 1996). The homogenates were incubated with α-amylase (Sigma-Aldrich) at a final concentration of 25 mg/mL for 1 h at 37°C with shaking at 40 rpm. For concentration of viral particles, derived from digestive diverticula, the method provided by the Japanese Committee for Standardization Virus of Detection in Food (2010) was used. Briefly, 20 min after centrifugation at 8000 g, the supernatant was recovered. To concentrate the virus, polyethylene glycol 6000 (Sigma-Aldrich) and sodium chloride (Wako) were added to the final concentration at 8% and 2.1%, respectively. Eighteen hours after incubation at 4°C, the supernatant was centrifuged at 8000 g for 20 min. After removing the supernatant, the pellet was resuspended in 200 μL of SDS-Tris-glycine buffer (containing 2.5 mM Tris, 19.2 mM glycine, 0.01% SDS, and pH 8.3) (BIO RAD). The resulting virus suspension was used for RNA extraction.

RNA extraction and reverse transcription

Viral RNA was extracted from 200 μL of viral suspension using a High Pure Viral RNA Kit (Roche diagnostics) with recombinant DNase I (Roche diagnostics). First strand cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kits (Life Technologies), according to the manufacturer's instructions.

NGS analysis of the capsid N/S region of VP1 gene by Illumina MiSeq

To amplify the partial capsid N/S region of noroviruses by RT-PCR, primers were prepared as shown in Table 1. An Applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystem) was used, and the PCR protocol was as follows. An initial incubation for 3 min at 94°C was followed by 40 cycles of 94°C for 60 s, 50°C for 60 s, and 72°C for 2 min, with an additional 15-min elongation step at 72°C after the last cycle. This PCR procedure was repeated using internal primers as nested PCR. Following the purification of the products using AMPure XP beads (Beckman Coulter), amplification and concentration of the nested-PCR products were determined by an Agilent 2200 TapeStation System (Agilent Technologies Japan Ltd.). Amplicons were diluted and pooled to generate a mixture containing an equimolar representation of each sample, used for one plate for sequencing, and then purified using a QIAquick PCR Purification Kit (Qiagen). Libraries were prepared using TruSeq ChIP Sample Prep Kit (Illumina K.K.) and were sequenced using an Illumina MiSeq (Illumina K.K.) with 300-base paired-end reads to ensure that the average number of reads per sample ranged from 200,000 to 400,000 (96 samples/RUN). All kits described above were used according to the manufacturer's instructions. Consequently, average length of bases obtained/sample was 80 Mbp (min: 11 Mbp; max: 285 Mbp). Average number of reads obtained/sample was 264,806 reads (min: 36,842; max: 948,138).

Table 1.

List of Primers to Amplify the Partial Capsid N/S Region of Noroviruses

Genogroup			Sense	Sequence (5′-3′) ^a,b	Product size (bp) ^c	Reference
GI	1st PCR	COG1F	+	CGYTGG ATG CGN TTY CAT GA	381	Kageyama et al. (2003)
		G1-SKR	−	CCA ACC CAR CCATTR TAC A		Yan et al. (2003)
	2nd PCR	G1-SKF	+	[Adaptor-A]+CTG CCC GAA TTY GTA AAT GA	330	Kageyama et al. (2003)
		G1-SKR	−	[Adaptor-B]+CCA ACC CAR CCA TTR TAC A		Yan et al. (2003)
GII	1st PCR	COG2F	+	CAR GAR BCN ATG TTY AGRTGG ATG AG	387	Kageyama et al. (2003)
		G2-SKR	−	CCR CCN GCA TRH CCR TTR TAC AT		Yan et al. (2003)
	2nd PCR	G2-SKF	+	[Adaptor-A]+CNT GGG AGG GCG ATC GCA A	344	Yan et al. (2003)
		G2-SKR	−	[Adaptor-B]+CCR CCN GCA TRH CCR TTR TAC AT		Yan et al. (2003)

Y = C + T; R = A + G; M = A + C; D = A + T + G; W = A + T.

Sequence of [Adaptor-A] was 5′-AATGATACGGCGACCACCGAGATCTACACxxxxxxxxACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′.

Sequence of [Adaptor-B] was 5′-CAAGCAGAAGACGGCATACGAGCATACGAGATyyyyyyyyGTGACTGGAGTTCAGGACGTGTGCTCTTCCGATCT-3′.

Index (barcode) sequence to identify each sample was presented as xxxxxxxx/yyyyyyyyy in the adaptor.

Adaptor was not included in the product size.

Read mapping for genotyping of noroviruses

The paired-end reads from MiSeq were also adaptor and quality trimmed, and read pairs were assembled into consensus sequences using FastqJoin version 1.1.2-806 (http://code.google.com/p/ea-utils/wiki/FastqJoin). Resultant reads were mapped against reference sequences using Burrows-Wheeler Aligner version 0.7.10 (http://bio-bwa.sourceforge.net/), and output alignment data were sorted using SAMtools version 1.2 (www.htslib.org/man/samtools). Counting reads mapped against each reference sequence were performed by custom script. The GenBank accession numbers of genes for read mappings in the article are listed in Table 2 (Kroneman et al., 2013).

Table 2.

Norovirus Capsid Sequences in This Study

Genotype no.	Strain ID	GenBank no.
GI.1	Norwalk/68/US	M87661
GI.2	Southampton/91/UK	L07418
GI.3	DesertShield/90/US	U04469
GI.4	Chiba407/87/JP	AB042808
GI.5	Musgrove/89/UK	AJ277614
GI.6	Hesse(BS5)/98/GE	AF093797
GI.7	Winchester/94/UK	AJ277609
GI.8	Boxer/01/US	AF538679
GI.9	Vancouver730/2004/CA	HQ637267
GII.1	Hawaii	U07611
GII.2	Melksham	X81879
GII.3	Tronto	U02030
GII.4	Bristol	X76716
GII.5	Hillingdon/90/UK	AJ277607
GII.6	Seacroft/90/UK	AJ277620
GII.7	Leeds/90/UK	AJ277608
GII.8	Amsterdam	AF195848
GII.9	VA97207/97	AY038599
GII.10	Erfurt/546/00/DE	AF427118
GII.11	SwNoV/Sw918/97/JP	AB074893
GII.12	Wortley/90/UK	AJ277618
GII.13	Fayettevil/98/US	AY113106
GII.14	M7/99/US	AY130761
GII.15	J23/1999/US	AY130762
GII.16	Triffin/1995/US	AY502010
GII.17	CSE1/2002/US	AY502009
GII.18	SwNoV/OHQW101/2003/US	AY823304
GII.19	SwNoV/OHQW170/2003/US	AY823306
GII.20	Luckenwalde591/2002/DE	EU373815
GII.21	IFI1998/2003/IR	AY675554
GII.22	YURI/JP	AB083780

To calculate the proportion of each genotype, the number of reads mapped against each reference was divided by the total reads obtained.

Statistical analysis

Significant differences for detection rate and proportion of genotypes were determined using the Fisher's exact test and the Mann–Whitney U test, respectively. Significant differences were defined as p < 0.01.

Results

Elimination of bacteria from oysters through depuration

To confirm operational status of the depuration procedure, APC, MPN of Escherichia coli and V. parahaemolyticus in oysters, was investigated. As shown in Table 3, four of nine and five of nine pre-depuration samples were under the limit of quantification (LOQ) for APC and fecal coliforms, respectively. In contrast, eight of nine and nine of nine post-depuration samples were under the LOQ for APC and fecal coliforms, respectively. V. parahaemolyticus was not detected in either the pre- or post-depurated samples. Total coliforms in seawater were not confirmed from poststerilized samples. However, three of nine presterilized samples were over the LOQ for total coliforms.

Table 3.

Elimination of Bacteria from Oyster Through Depuration

		APC (cfu/g) ^a		Fecal coliforms (MPN/100 g) ^b		V. parahaemolyticus (MPN/g) ^c		Total coliforms (MPN/g) ^d
Day of sampling	Sample	Pre^e	Post^e	Pre	Post	Pre	Post	Pre	Post
January 5, 15	Oysters	LOQ	LOQ	20	LOQ	LOQ	LOQ	—	—
	Seawater	—	—	—	—	—	—	4.5	LOQ
January 12, 15	Oysters	LOQ	LOQ	LOQ	LOQ	LOQ	LOQ	—	—
	Seawater	—	—	—	—	—	—	LOQ	LOQ
January 19, 15	Oysters	2200	LOQ	LOQ	LOQ	LOQ	LOQ	—	—
	Seawater	—	—	—	—	—	—	LOQ	LOQ
January 26, 15	Oysters	470	LOQ	LOQ	LOQ	LOQ	LOQ	—	—
	Seawater	—	—	—	—	—	—	2	LOQ
February 2, 15	Oysters	350	LOQ	20	LOQ	LOQ	LOQ	—	—
	Seawater	—	—	—	—	—	—	LOQ	LOQ
February 9, 15	Oysters	LOQ	LOQ	LOQ	LOQ	LOQ	LOQ	—	—
	Seawater	—	—	—	—	—	—	LOQ	LOQ
February 16, 15	Oysters	LOQ	LOQ	LOQ	LOQ	LOQ	LOQ	—	—
	Seawater	—	—	—	—	—	—	LOQ	LOQ
February 23, 15	Oysters	360	LOQ	20	LOQ	LOQ	LOQ	—	—
	Seawater	—	—	—	—	—	—	LOQ	LOQ
March 2, 15	Oysters	3700	760	45	LOQ	LOQ	LOQ	—	—
	Seawater	—	—	—	—	—	—	2	LOQ

Limit of quantification for the total aerobic bacteria is 300 cfu/g.

Limit of quantification for the fecal coliforms is 18 MPN/100 g.

Limit of quantification for V. parahaemolyticus is 3 MPN/g.

Limit of quantification for the total coliforms is 1.8 MPN/100 mL.

Pre- and Post-depuration were expressed as “Pre” and “Post,” respectively.

APC, aerobic plate count; LOQ, limit of quantification; MPN, most probable number.

Profiling of norovirus GI genotypes by NGS

As shown in Table 4, norovirus GI was not detected in any pre-depurated samples. In contrast, VP1 region derived from GI was detected from eight samples of post-depurated C. gigas. However, depuration resulted in no significant changes in either the detection rate or proportion of norovirus GI genotypes.

Table 4.

Elimination Efficacy of Norovirus GI Genotypes

	Number of positive samples (%) ^a,b		Proportion % ^a –c
Geno-type	Pre (n = 89)	Post (n = 88)	Pre (n = 0)	Post (n = 8)
GI.1	ND	3 (3.41)	ND	2.77
GI.2	ND	8 (9.09)	ND	30.78
GI.3	ND	8 (9.09)	ND	0.04
GI.4	ND	8 (9.09)	ND	62.46
GI.5	ND	ND	ND	ND
GI.6	ND	4 (4.55)	ND	2.76
GI.7	ND	ND	ND	ND
GI.8	ND	2 (2.27)	ND	≤0.01
GI.9	ND	ND	ND	ND

Pre- and Post-depuration were expressed as “Pre” and “Post,” respectively.

ND was used as the abbreviation for “not detected.”

Relative abundance rate less than 0.01 represents as “≤0.01.”

Profiling of norovirus GII genotypes by NGS

VP1 region derived from GII was detected from 77 and 78 samples of pre- and post-depurated C. gigas, respectively. As shown in Table 5, GII.1 to 6, GII.13, GII.14, and GII.16 to19 were detected from pre-depurated samples and the detection rate differed depending on the norovirus genotype. Similarly, GII.1 to 6, GII.9, GII.11, GII.13, GII.14, GII.16 to 19, and GII.21 were detected in post-depurated samples and the detection rate also differed depending on the norovirus genotype. However, the depuration procedure resulted in no significant changes in either the detection rate or the proportion of norovirus GII genotypes. The detection rate and proportion observed were highest for GII.17.

Table 5.

Elimination Efficacy of Norovirus GII Genotypes

	Number of positive samples (%) ^a,b		Proportion % ^a –c
Genotype	Pre (n = 89)	Post (n = 88)	Pre (n = 77)	Post (n = 78)
GII.1	6 (6.74)	2 (2.27)	≤0.01	≤0.01
GII.2	1 (1.12)	1 (1.14)	≤0.01	≤0.01
GII.3	75 (84.27)	74 (84.09)	1.95	2.07
GII.4	62 (69.66)	61 (69.32)	0.58	0.99
GII.5	1 (1.12)	1 (1.14)	≤0.01	≤0.01
GII.6	1 (1.12)	8 (9.09)	≤0.01	≤0.01
GII.7	ND	ND	ND	ND
GII.8	ND	ND	ND	ND
GII.9	ND	1 (1.14)	ND	≤0.01
GII.10	ND	ND	ND	ND
GII.11	ND	2 (2.27)	ND	≤0.01
GII.12	ND	ND	ND	ND
GII.13	71 (79.78)	68 (77.27)	0.95	1.27
GII.14	6 (6.74)	1 (1.14)	0.03	≤0.01
GII.15	ND	ND	ND	ND
GII.16	52 (58.43)	49 (55.68)	≤0.01	≤0.01
GII.17	77 (86.52)	78 (88.64)	95.98	95.14
GII.18	57 (64.04)	65 (73.86)	0.50	0.50
GII.19	5 (5.62)	6 (6.82)	≤0.01	≤0.01
GII.20	ND	ND	ND	ND
GII.21	ND	4 (4.55)	ND	≤0.01
GII.22	ND	ND	ND	ND

Pre- and Post-depuration were expressed as “Pre” and “Post,” respectively.

ND was used as the abbreviation for “not detected.”

Relative abundance rate less than 0.01 represents as “≤0.01.”

Discussion

Before evaluating profile of noroviruses in pre- and post-depurated oysters, we first confirmed operational status of the applied depuration procedure by monitoring bacterial agents in oysters. Results indicated that the applied depuration procedure was able to reduce the number of bacterial agents to a level (APC less than 50,000 cfu/g, fecal coliforms less than 230 MPN/100 g, and V. parahaemolyticus less than 100 MPN/g) recommended by the Japanese Ministry of Health, Labour and Welfare (JMHLW, 1967). This suggests that the procedure may be recommended to improve sanitation levels in oysters used for consumption. In this study, we did not consider the conditions of depuration, except for the sterilization of seawater by UV light. This reflected an effort to investigate depuration procedures used in our country. Generally, efficacy of depuration, in regards to bacterial agents, appears to vary with conditions of the procedure (Souza et al., 2013). Specifically, temperature and salinity have been well studied for their effect on depuration (Ueki et al., 2007; Larsen et al., 2013). For environments highly contaminated by bacteria, finding effective conditions of depuration is expected to protect consumers.

The persistence of norovirus in oysters after depuration could be attributed to specific virion binding with surface carbohydrates expressed in oyster tissues, as described by Le Guyader et al. (2006). Genogroup-specific differences have been reported with regard to sensitivity to removal and binding to receptors (Maalouf et al., 2010; Tuladhar et al., 2012). If depuration efficacy differed among norovirus genotypes, the proportion of norovirus genotypes, present in oysters, was hypothesized to change after depuration. Thus, to investigate depuration efficacy on each genotype, the detection rate and proportion of norovirus genotypes, in pre- and post-depurated oysters, were determined using NGS analysis. As a result, no significant differences in both the detection rate and proportion of norovirus genotypes were observed. In addition, it was suggested that depuration might not have the ability to remove a specific population of noroviruses from oysters. Previous studies, using quantitative RT-PCR to demonstrate the persistence of noroviruses in oysters, also support the current results and suggest that reducing the concentration of noroviruses by depuration is difficult (Schwab et al., 1998; Ueki et al., 2007; Savini et al., 2009). Certain norovirus genotypes were detected in post-depuration samples, despite the absence of such norovirus genotypes in the pre-depuration samples. This discrepancy may be explained by the seawater used for depuration. The seawater taken for the depuration was not identical to the seawater in which oysters were aquacultured. However, it is still very difficult to quantify the amount of noroviruses directly from seawater. Further studies are needed to discuss this phenomenon.

Although information on the epidemiological characteristics of noroviruses in oysters has been limited so far, this study showed the diversity of norovirus genotypes in the oyster. Oysters appeared to harbor various norovirus genotypes and the detection rate and proportion in oysters differed among genotypes. This phenomenon may be explained by strain-dependent norovirus bioaccumulation in oysters reported by Maalouf et al. (2011). For example, bioaccumulation of GI.1 specifically occurred in digestive tissues in a dose-dependent manner and efficiency paralleled ligand expression in the oyster, which was highest during cold months (Maalouf et al., 2011). In comparison, GII.4 displayed poor bioaccumulation and was recovered in almost all tissues without seasonal influence (Maalouf et al., 2011). Thus, on the assumption that norovirus strain levels in the surrounding environment of the investigated oyster remain unchanged, the relationship between the ligand expressed in oysters and the corresponding receptor on the norovirus particle may affect the rate of detection and proportion of each norovirus genotype in the oyster.

Among various genotypes observed in the present study, GII.17 was predominant and will be discussed because of its recent attention from a public health perspective. Frequent detection of GII.17 from oysters may reflect the true virus circulation resulting from cases of infectious gastroenteritis. In fact, genetic analyses of GII.17 in diarrheal disease outbreaks from December 2014 to March 2015 in Japan demonstrated that a novel norovirus variant, GII.P17-GII.17, was prevalent in Japan from December 2014 onward, and GII.17 has been the predominant variant beyond GII.4 (Matsushima et al., 2015). Thus, JMHLW has advised that the GII.17 variant, which was first confirmed from clinical specimen sampling during 2014 in Japan, is a candidate genotype for the next major outbreak in Japan (JMHLW, 2015).

Conclusions

To our knowledge, this is the first report to evaluate depuration efficacy on reducing norovirus levels, using NGS, and to demonstrate the profiles of noroviruses in the pre- and post-depurated oysters. The NGS technique applied in this study seems to have great power to investigate the proportion of norovirus strains in oysters, as we were able to detect norovirus genotypes with a proportion less than 0.0001%. Although the conventional oyster depuration procedure is not currently sufficient for the removal of noroviruses, this procedure appears to be applicable for decontamination of bacterial agents. Further studies are needed to design effective protocols for removal and/or inactivation of noroviruses from contaminated oysters. In addition, a better understanding of the relationship between noroviruses and oysters, in terms of contamination, pathogen persistence, and pathogen selection, may help improve the sanitary level of shellfish through the development of new methods to prevent oyster contamination or to depurate contaminated oysters.

Footnotes

Acknowledgment

This study was performed as part of the surveillance/monitoring program of microbiological hazards in the Ministry of Agriculture, Forestry and Fisheries, Tokyo, Japan.

Disclosure Statement

No competing financial interests exist.

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