Effect of High Pressure Processing on a Wide Variety of Human Noroviruses Naturally Present in Aqua-Cultured Japanese Oysters

Abstract

The contamination of oysters with human norovirus (HuNoV) poses a human health risk, as oysters are often consumed raw. In this study, the effect of high pressure processing (HPP) on a wide variety of HuNoVs naturally present in aqua-cultured Japanese oysters was determined through a polymerase chain reaction-based method with enzymatic pretreatment, to distinguish between infectious HuNoV. Among five batches, genogroup I. genotype 1 (GI.1), GI.2, GI.3, and GI.8 HuNoV were detected from only one oyster not treated with HPP in the fifth batch, while genogroup II. genotype 1 to 4 (GII.1 to 4), GII.6, GII.8., GII.9, GII.13, GII.16, GII.17, and GII.22 HuNoV were detected from oysters not treated with HPP in all tested batches as determined by next-generation sequencing analysis. Neither GI nor GII HuNoV was detected in the oysters of any of the batches after HPP treatment. To our knowledge, this is the first study to investigate the effect of HPP on a wide variety of HuNoVs naturally present in aqua-cultured oysters.

Introduction

Human norovirus (HuNoV) infection via consumption of HuNoV-contaminated shellfish is a worldwide problem (Kingsley et al., 2002; Le Guyader et al., 2006; JEMRA, 2008; Noda et al., 2008), leading to not only severe economic loss for the industry due to product-recalls and farm closures but also the loss of consumer confidence (Morse et al., 1986; Richards, 2006). Thus, there is a need for an intervention to reduce the outbreaks of foodborne diseases resulting from the consumption of raw oysters contaminated with HuNoV.

HuNoV belongs to the family Caliciviridae under the genus Norovirus, which is divided into seven genogroups numbered genogroup I (GI) to GVII, based on their genome sequence similarity (Rocha-Pereira et al., 2016). Two genogroups (GI and GII) are known mainly to infect humans. There are 9 and 22 genotypes assigned with GI and GII genogroups, respectively (Kroneman et al., 2013). Because different strains of HuNoV can differ in their incidence, virulence, stability, and ability to cause epidemics (Fankhauser et al., 2002; Verhoef et al., 2009; Huhti et al., 2011; Tuladhar et al., 2012; Kroneman et al., 2013), it is important to correctly identify genetically different HuNoV for evaluating the efficacy of control measures.

Recently, high pressure processing (HPP) has been identified as a promising nonthermal intervention to treat high-risk food for virus contamination without significant changes in its nature (López-Caballero et al., 2000). When oyster homogenate, contaminated by inoculation of NoV genogroup II.4 (GII.4), was treated at 300 MPa for 5 min at 6°C, a 3.51 log₁₀ reduction in the HuNoV genome was observed (Ye et al., 2014). When oysters were contaminated by an injection of GI.1 HuNoV into the digestive tract, and treated at 600 MPa for 5 min at 6°C, no HuNoV infection was observed in clinical trials (Leon et al., 2011). However, direct assessments of the effects of HPP on HuNoV naturally present in oysters have been performed limited so far. It should be taken into account that the aqua-cultured oyster bio-accumulates a wide range of HuNoV as determined by next-generation sequencing (NGS) analysis (Imamura et al., 2016a, b, 2017a). The validity of using HPP to a reduction of a wide range of HuNoV strains is likely to be promising, because Ye et al. (2014) demonstrated susceptibility of different HuNoV strains to HPP using the porcine gastric mucin magnetic beads assay.

Meanwhile, replication of HuNoV in stem cell-derived human enteroids reported by Ettayebi et al. (2016) will hopefully applied for assessing the level of inactivation of HuNoV in food. However, the in vitro cell culture system has not been routinely available yet. Currently, reverse transcription (RT)–polymerase chain reaction (PCR) is the most popular detection method for HuNoV in food analysis. The practical disadvantage of the method is that it is unable to distinguish between infectious and noninfectious viruses (Baert et al., 2008; Lowther et al., 2010). Thus, several researchers have proposed enzymatic pretreatment of the sample by RNase before RT reaction to digest viral RNA-derived noninfectious virus particles (Nuanualsuwan and Cliver, 2002; Baert et al., 2008; Lamhoujeb et al., 2008). This has already been applied to the evaluation of inactivation, when studying HuNoV responses to various interventions (Baert et al., 2008; Topping et al., 2009; Ye et al., 2014).

Thus, this study aimed to analyze the survival of a variety of HuNoVs in aqua-cultured oysters after HPP treatment using combined enzymatic pretreatment and NGS analysis.

Materials and Methods

Oysters

Sixty shell-on oysters (Crassostrea gigas) per batch were purchased from the Momonoura producer of oyster consolidated company at Miyagi prefecture in Japan, from January to February 2017.

HPP of oysters

HPP was conducted at the Momonoura producer of oyster consolidated company. Sixty shell-on oysters per batch came from the same aquaculture environment (all under same conditions) were divided into 2 groups of 30 shell-on oysters each, and one of the groups was treated by HPP at 400 MPa for 5 min at around 10°C, which corresponds to the temperature of seawater in the aquaculture area. Each group of oysters was heat-sealed using the Impulse food sealer. The experiment was repeated five times. In total, 150 shell-on oysters treated with HPP and 150 shell-on oysters not treated with HPP were subjected to the current study. Oysters were refrigerated and shipped overnight to the Incorporated Foundation Kenbikyo-in for HuNoV detection and quantification.

Sample preparation of viral suspensions and enzymatic pretreatment

For the preparation of viral suspensions, the digestive diverticula were collected by dissection on the day of arrival to the laboratory. The sample was homogenized in nine times its volume of phosphate-buffered saline (PBS) solution (without magnesium and calcium). The homogenates were incubated with α-amylase (Wako, Tokyo, Japan) at a final concentration of 2.5 mg/mL for 1 h at 37°C, and vortexed every 15 min. To concentrate the viral particles derived from digestive diverticula, the method provided by the Japanese Committee for Standardization Virus of Detection in Food (2010) was used. After centrifugation at 8000 × g for 20 min, the supernatant was recovered. To concentrate the virus, polyethylene glycol 6000 (Wako) and sodium chloride (Wako) were added at a final concentration of 12% and 5.8%, respectively. Eighteen hours after incubation at 4°C, the samples were centrifuged at 8000 × g for 20 min. After removing the supernatant, the pellet was resuspended in 400 μL of PBS (-) (containing 0.5% Zwittergent 3–14 Detergent) (Merk, Frankfurt, Germany). Fourteen units (1.4 μL) of RNase ONE™ ribonuclease (Promega, WI), 10 μL of 10 × reaction mixture, and 18.6 μL of the distilled water were added to 70 μL of the resulting virus suspension, and the mixture (total volume of 100 μL) was incubated at 37°C for 1 h (Topping et al., 2009). The resulting enzyme-treated virus suspension was added to 100 μL of PBS (-), and used for RNA extraction.

RNA extraction and RT

Viral RNA was extracted from 200 μL of viral suspension using a High Pure Viral RNA kit (Roche Diagnostics, Tokyo, Japan) with recombinant DNase I (Roche Diagnostics), according to the manufacturer's instructions with slight modifications. To promote the recovery of RNA, 8 μL of MS2 RNA (Roche Diagnostics) was added to 400 μL of the binding buffer. cDNA synthesis of the first strand was performed using a High-Capacity cDNA Reverse Transcription kit (Life technologies, Tokyo, Japan). To confirm presence of RNAs in the sample, resulting RNA solutions were subjected to agarose 1% gel electrophoresis. To avoid detection of reverse transcripts due to the fragmented genome (and thus noninfective), Oligo dT primer was used for RT.

Detection of the capsid N-terminal shell (N/S) region of the VP1 gene

To amplify the partial capsid N/S region of HuNoV by RT-PCR, primers were prepared as shown in Table 1. An Applied Biosystems Veriti 96 Well Thermal Cycler (Applied Biosystems, CA) was used, and the PCR protocol was as follows: an initial incubation for 15 min at 95°C, followed by 40 cycles at 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 for nested PCR. Aliquots (8 μL) of the PCR products were analyzed by 2% agarose gel electrophoresis using 1 μg/mL ethidium bromide. The theoretical limit of detection is 228.6 copies per 1 g of digestive diverticula, based on the assumption that the recovery rate is 100%.

Table 1.

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

Genogroup			Sense	Sequence (5′–3′) ^a	Product size (bp)	Reference
GI	First 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)
	Second PCR	G1-SKF	+	CTG CCC GAA TTY GTA AAT GA	330	Kageyama et al. (2003)
		G1-SKR	−	CCA ACC CAR CCA TTR TAC A		Yan et al. (2003)
GII	First 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)
	Second PCR	G2-SKF	+	CNT GGG AGG GCG ATC GCA A	344	Yan et al. (2003)
		G2-SKR	−	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.

N/S, N-terminal shell; PCR, polymerase chain reaction.

Quantification of HuNoV by real-time PCR

Samples that tested positive for the partial capsid N/S region of HuNoV by RT-PCR were processed using real-time PCR to quantify HuNoV capsid genes using the TaKaRa qPCR Norovirus (GI/GII) Typing Kit (Takara, Tokyo, Japan), according to the manufacturer's instructions. All amplification was performed in duplicate. When the R ² value was over 0.990, the real-time PCR procedure was regarded as successful, and the samples were analyzed.

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

Pimers were prepared as shown in Table 1. The PCR protocol was carried out in an Applied Biosystems Veriti 96 Well Thermal Cycler (Applied Biosystems) as follows: an initial incubation for 3 min at 94°C, followed by 40 cycles at 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 with an adaptor sequence for MiSeq sequencing by nested PCR. Following the purification and amplification of the PCR products using AMPure XP beads (Beckman Coulter, CA), the concentration of the nested-PCR products was determined using an Agilent 2200 TapeStation System (Agilent Technologies Japan Ltd., Tokyo, Japan). Amplicons were diluted and pooled to generate a mixture containing an equimolar representation of each sample. Pooled amplicons were purified using a QIAquick PCR Purification Kit (Qiagen, CA), and sequenced using the 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 the kits were used according to the manufacturer's instructions. Consequently, the average length of reads obtained per sample was 110 Mbp (min; 71 Mbp: max; 140 Mbp). The average number of reads obtained per sample was 364,347 reads (min; 234,834: max; 466,618).

Read mapping for genotyping of HuNoV

The paired-end reads from MiSeq were 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). The number of reads mapped against each reference sequence was counted with a custom script. The GenBank accession numbers of genes for read mappings used in this article are in accordance with those mentioned in the previous reports (Imamura et al., 2016a, b, 2017a).

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 in the detection rate and proportion of genotypes were determined using the Fisher's exact test. Significant differences were defined for all values of p < 0.01.

Results

Detection and quantification of GI in oysters treated with/without HPP

Table 2 provides details about the detection rate of HuNoV in natural oysters treated with/without HPP in each tested batch. HuNoV GI was detected in only one oyster not treated with HPP in the fifth batch as determined by RT-PCR, and the genome copy number in the sample was quantified as 3.4 × 10² copies/g as determined by real-time PCR. In contrast, GI was not detected in oysters treated with HPP.

Table 2.

Detection and Quantification of Human Norovirus Naturally Present in Oysters Treated With/Without High Pressure Processing

				GI			GII
Batch	Date of sampling	Treatment	n	No. of positive	%	Genome copy no/g of digestive diverticula ^a	No. of positive	%	Genome copy no/g of digestive diverticula ^a
First	January 11	—	30	0	0		1	3.3	5.0 × 10²
		HPP	30	0	0		0	0
Second	January 18	—	30	0	0		6	20.0	9.0 × 10²
		HPP	30	0	0		0	0^b
Third	January 26	—	30	0	0		7	23.3	6.2 × 10²
		HPP	30	0	0		0	0^b
Fourth	February 1	—	30	0	0		6	20.0	4.3 × 10²
		HPP	30	0	0		0	0^b
Fifth	February 22	—	30	1	3.3	3.4 × 10²	11	36.6	4.9 × 10²
		HPP	30	0	0		0	0^b

Average of positive sample.

Asterisk demonstrates significant difference of detection rate between groups treated with/without HPP (p < 0.01).

HPP, high pressure processing.

Detection and quantification of GII in oysters treated with/without HPP

HuNoV GII was detected in oysters not treated with HPP in all tested batches, while the detection rates were different between the batches. The lowest detection rate was observed in the first batch, whereas the highest was in the fifth batch. The average genome copy number in oysters not treated with HPP in the first, second, third, fourth, and fifth batches was 5.0 × 10², 9.0 × 10² (range: 3.0 × 10² to 2.4 × 10³), 6.2 × 10² (range: 2.3 × 10² to 2.0 × 10³), 4.3 × 10² (range: 3.0 × 10² to 7.1 × 10²), and 4.9 × 10² (range: 2.3 × 10² to 1.2 × 10³), respectively (Table 2). In contrast, HuNoV GII was not detected in any of the oysters treated with HPP in any of the batches. There were significant differences in the detection rate of HuNoV GII between oysters treated with/without HPP in each batch except for the first batch (p < 0.01).

Profiles of HuNoV genotypes present in oysters not treated with HPP

NGS revealed a wide range of HuNoV genotypes present in oysters not treated with HPP (Table 3). GI.2, GI.3, and GI.8 HuNoV were detected from a sample positive for GI. GII.1−4, GII.6, GII.8, GII.9, GII.13, GII.16, GII.17, and GII.22 were detected from a sample positive for GII. The detection rates and proportions of detected genotypes differed depending on the sampling period. In total, the detection rates of GII.2 and GII.17 were highest among all genotypes. GII.2 was found in the highest proportion in oysters (99.96%, 66.67%, 57.18%, 50.04%, and 61.57% for the first to fifth batch, respectively), followed by GII.17 (≤0.01%, 33.32%, 28.57%, 33.32%, and 29.35% for the first to fifth batch, respectively).

Table 3.

Detection of Human Norovirus Genotypes Present in Japanese Oysters Treated Without High Pressure Processing

Batch
	Genogroups		Number of positive for GI/GII ^a
		Genotypes ^b		Number of positive sample ^c
First
	GII		1
		GII.2		1
		GII.3		1
		GII.4		1
		GII.13		1
		GII.17		1
Second
	GII		6
		GII.2		6
		GII.3		6
		GII.4		2
		GII.6		2
		GII.13		6
		GII.16		5
		GII.17		6
Third
	GII		7
		GII.1		1
		GII.2		7
		GII.3		2
		GII.6		2
		GII.13		3
		GII.16		6
		GII.17		7
		GII.22		2
Fourth
	GII		6
		GII.1		1
		GII.2		6
		GII.3		5
		GII.6		4
		GII.9		1
		GII.13		2
		GII.16		6
		GII.17		6
		GII.22		1
Fifth
	GI		1
		GI.2		1
		GI.3		1
		GI.8		1
	GII		11
		GII.1		1
		GII.2		11
		GII.3		5
		GII.6		6
		GII.13		5
		GII.16		7
		GII.17		11

Number of GI/GII-positive samples were demonstrated.

Genotypes not detected in this study were abbreviated.

Number of positive samples for each genotype were demonstrated.

Discussion

For the detection of infectious HuNoV, Nuanualsuwan and Cliver (2002) originally used protease K (PK) followed by RNase treatment to show that damaged virus particles can be rendered undetectable by RT-PCR. A previous study considering the necessity of PK treatment done by Noda (2014) demonstrated that no significant difference was observed between simultaneous treatment of PK with RNase and RNase alone. Thus, we used RNase alone for pretreatment during sample preparation. We used α-amylase to reduce an influence of PCR inhibitor in the oyster sample but not replacement for PK treatment, since treatment of α-amylase have been reported to improve detection of HuNoV gene by RT-PCR (Noda et al., 2006).

Variable sensitivities of HuNoV to HPP were reported in several studies. For example, Lou et al. (2016) demonstrated that the resistance of HuNoV genotypes to HPP ranked as follows: GII.1 > GII.6 > GII.4. The key question to be answered in this study was whether HPP was effective against a greater variety of HuNoVs naturally present in aqua-cultured oysters. The HuNoV identified in 30 oysters not treated with HPP were of 3 GI genotypes, 9 GII genotypes as determined by NGS, suggesting that these genotypes may be susceptible to HPP at 400 MPa for 5 min, given that another 30 oysters treated with HPP were assumed to have the same viral load and diversity. However, further studies are needed to clarify the susceptibility of HuNoV with different strains to HPP, by testing the sample prepared by spiking homogenate digestive tissues of aqua-cultured oysters. In addition, an extraction control was not used in this study and therefore there is possibility that HuNoV present in the oyster were not successfully recovered.

In a previous study, a 1.87 log₁₀ and 1.99 log₁₀ reduction of GII.17 genome concentration in laboratory-contaminated oysters was observed after HPP at 400 MPa for 5 min at 25°C (Imamura et al., 2017b). Thus, we suppose that the levels of HuNoV in commercial oysters may be reduced by HPP at 400 MPa for 5 min to an undetectable level when determined by RT-PCR. In fact, concentrations of HuNoV in oysters not treated with HPP were less than 1000 copies/g, and the HuNoV genome was not detected in the samples prepared from oysters treated with HPP at 400 MPa for 5 min at around 10°C, suggesting that HPP successfully reduced infectious HuNoV in oysters to a level under the detection limit of RT-PCR. However, it needs to be addressed that the theoretical limit of detection of the method is probably much higher because 100% recovery is unattainable.

We need to consider whether the level of HuNoV concentration under the detection limit of RT-PCR directly corresponds to the level needed to protect humans from HuNoV infection. Lowther et al. (2010) demonstrated that the genomic mean of the levels in outbreak samples was 1048 copies/g, and none of the outbreak-related samples contained fewer than 152 copies/g, suggesting that the undetectable level of HuNoV by RT-PCR may also be the level that does not cause oyster-related gastroenteritis. Thus, we suppose that the levels of HuNoV in commercial oysters may be reduced by HPP at 400 MPa for 5 min to the level that might not cause oyster-related gastroenteritis. However, HPP at 300 MPa or below, applied at environmental temperature is used by the shellfish industry for facilitating oyster shucking, extending shelf life, and reducing Vibrio spp (Ye et al., 2012). Thus, the HPP conditions utilized in this study may not be accepted by the industries. Further studies are needed to determine the optimum conditions for processing aqua-cultured oysters while using as low a pressure as possible. Meanwhile, Kingsley et al. (2014) demonstrated that organoleptic changes in sterile triploid oysters induced by HPP at different temperatures and elevated pressures would be accepted by consumers. Furthermore, Kingsley et al. (2015) also proposed that flavoring a raw oyster by HPP provides the potential to create a microbiologically safe product with unique sensory characteristics, which may influence consumer acceptance and marketability.

Conclusions

To our knowledge, this is the first report to investigate the effect of HPP on a wide variety of HuNoVs naturally present in oysters. However, detailed understanding of the level of HPP inactivation on each genotype and the treatment conditions on HuNoV naturally present in oysters needs to be considered.

Footnotes

Acknowledgments

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

Disclosure Statement

No competing financial interests exist.

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