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Journal of Clinical Microbiology, December 2007, p. 3996-4005, Vol. 45, No. 12
0095-1137/07/$08.00+0     doi:10.1128/JCM.01516-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Norovirus Infections in Symptomatic and Asymptomatic Food Handlers in Japan

Kazuhiro Ozawa,¹ Tomoichiro Oka,² Naokazu Takeda,² and Grant S. Hansman^2*

Chubu Food and Environmental Safety Center, Shizuoka,¹ Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan²

Received 29 July 2007/ Returned for modification 11 August 2007/ Accepted 29 September 2007

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Noroviruses are the leading cause of outbreaks of gastroenteritis in the world. At present, norovirus genogroup II, genotype 4 (GII/4), strains are the most prevalent in many countries. In this study we investigated 55 outbreaks and 35 sporadic cases of norovirus-associated gastroenteritis in food handlers in food-catering settings between 10 November 2005 and 9 December 2006 in Japan. Stool specimens were collected from both symptomatic and asymptomatic individuals and were examined for norovirus by real-time reverse transcription-PCR; the results were then confirmed by sequence analysis. Norovirus was detected in 449 of 2,376 (19%) specimens. Four genogroup I (GI) genotypes and 12 GII genotypes, including one new GII genotype, were detected. The GII/4 sequences were predominant, accounting for 19 of 55 (35%) outbreaks and 16 of 35 (46%) sporadic cases. Our results also showed that a large number of asymptomatic food handlers were infected with norovirus GII/4 strains. Norovirus GII had a slightly higher mean viral load (1 log unit higher) than norovirus GI, i.e., 3.81 x 10⁸ versus 2.79 x 10⁷ copies/g of stool. Among norovirus GI strains, GI/4 had the highest mean viral load, whereas among GII strains, GII/4 had the highest mean viral load (2.02 x 10⁸ and 7.96 x 10⁹ copies/g of stool, respectively). Importantly, we found that asymptomatic individuals had mean viral loads similar to those of symptomatic individuals, which may account for the increased number of infections and the predominance of an asymptomatic transmission route.

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The positive-sense polyadenylated single-stranded RNA virus family Caliciviridae contains four genera: Norovirus, Sapovirus, Lagovirus, and Vesivirus (1). The prototype strain of human norovirus is the Norwalk virus (NV/Human/US/1968), which was first discovered in an outbreak of gastroenteritis in an elementary school in Norwalk, OH, in 1968 (15). Noroviruses are the leading cause of outbreaks of gastroenteritis in the world; they cause outbreaks in various settings, including hospitals, cruise ships, schools, and restaurants (2, 9, 12, 15, 23, 24, 29). In addition, noroviruses have been detected in environmental samples (e.g., treated and untreated sewage) as well as in contaminated foods such as oysters, shellfish, sandwiches, salads, raspberries, and even ice (7, 18, 19, 26). Numerous molecular epidemiological studies have revealed a global distribution of these viruses (25, 27, 31).

The most widely used method of detecting noroviruses is reverse transcription-PCR (RT-PCR), which has high sensitivity; also, the products can be used for further genetic analysis. Real-time RT-PCR assays have also been developed; they are sensitive, broadly reactive, and rapid for the detection of human noroviruses in clinical stool specimens and environmental samples (13, 14, 21).

As the detection methods become more and more sensitive, the numbers of genogroups and genotypes are expected to increase. One emerging characteristic is that strains have been found to persist in one geographical region, only to disappear suddenly (8, 10). Seasonal studies have commonly found norovirus outbreaks peaking in the winter periods; however, the incidence rates, detection rates, and overall prevalence rates of infections may differ by country and setting and are likely to be affected by diagnostic techniques.

Recently, noroviruses have been divided into five genetically distinct genogroups, but the majority of human noroviruses can be divided into two genetically distinct genogroups, genogroup I (GI) and GII, which can be subdivided into at least 14 GI and 17 GII genotypes (14). Norovirus genotype identities are generally maintained across the open reading frames (ORFs). However, a number of norovirus strains failed to maintain their sequence identities for RNA-dependent RNA polymerase and VP1, and they were shown to be recombinant (16, 20, 30). Evidence suggested that the recombination site occurred at the conserved polymerase and capsid junction between ORF1 and ORF2.

The purpose of this study was to investigate norovirus-associated gastroenteritis in food handlers at food-catering settings in Japan between 10 November 2005 and 9 December 2006. Four GI genotypes and 12 GII genotypes, including 1 new GII genotype, were detected. Our results showed that both symptomatic and asymptomatic food handlers were infected with noroviruses.

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Specimens. Fifty-five outbreaks and 35 sporadic cases of norovirus-associated gastroenteritis occurred at food-catering settings during the two winter periods between 10 November 2005 and 9 December 2006 (Fig. 1). Outbreaks were defined as having both (i) two or more food handlers with symptoms of gastroenteritis, i.e., nausea, vomiting, stomachache, diarrhea, or fever, and (ii) two or more specimens that were positive for norovirus by real-time RT-PCR. Sporadic cases were defined as having (i) only one symptomatic food handler and/or (ii) only one specimen positive for norovirus by real-time RT-PCR (even if two or more food handlers were symptomatic). Outbreak settings included nursing care centers, fast food establishments, hospitals, school canteens, hotels, restaurants, university cafeterias, and a kindergarten (see Tables 1 and 2). Stool specimens were collected from both symptomatic and asymptomatic employees. In total, 2,376 of approximately 7,000 specimens were examined for norovirus by real-time RT-PCR.

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FIG. 1. Map of Japan showing the prefectures/places where the specimens were collected.

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TABLE 1. Details of norovirus-associated gastroenteritis outbreaks in Japan

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TABLE 2. Details of sporadic cases of norovirus-associated gastroenteritis in Japan

Virus detection. A 10% (wt/vol) stool suspension was prepared with sterilized MilliQ water and centrifuged at 10,000 x g for 10 min. The QIAamp viral RNA minivacuum protocol (Qiagen, Hilden, Germany) was used to extract RNA from 140 µl of the clarified supernatant according to the manufacturer's instructions. Briefly, cDNA synthesis was carried out with 10 µl of the RNA in 20 µl of the reaction mixture containing 50 pmol random hexamer (Takara, Tokyo, Japan), 1x Superscript III reverse transcriptase buffer (Invitrogen, Carlsbad, CA), 10 mM dithiothreitol (Invitrogen), 0.4 mM each deoxynucleoside triphosphate (Roche, Mannheim, Germany), 1 U RNase inhibitor (Toyobo, Tokyo, Japan), and 10 U Superscript reverse transcriptase III (Invitrogen). RT was performed at 37°C for 15 min, followed by 50°C for 1 h. Real-time RT-PCR was performed as previously described, and the cutoff for positive norovirus specimens was set at >10 copies per well (13).

Sequencing and phylogenetic analysis. Conventional RT-PCR was carried out to sequence the real-time RT-PCR-positive specimens. Briefly, for norovirus GI PCR, primers G1SKF and G1SKR were used, and for norovirus GII PCR, primers G2SKF and G2SKR were used (17). RT-PCR products were excised from the gel and purified with the QIAquick gel extraction kit (Qiagen, Germany). Nucleotide sequences were prepared with the BigDye Terminator cycle sequencing kit (version 3.1) and determined with the ABI 3130 sequencer (ABI, Boston, MA). Nucleotide sequences were aligned with Clustal X, and the distances were calculated by Kimura's two-parameter method. Phylogenetic trees with bootstrap analysis from 1,000 replicas were generated by the neighbor-joining method as described previously (14).

Nucleotide sequence accession numbers. The accession numbers for sequences determined in this study are GenBank EF630426 to EF630534.

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Specimens and screening for norovirus by real-time RT-PCR. Fifty-five outbreaks and 35 sporadic cases of norovirus-associated gastroenteritis in Japan were examined between 10 November 2005 and 9 December 2006 (Tables 1 and 2; Fig. 1). Most outbreaks occurred at nursing care centers (25 of 55), followed by hospitals (12 of 55), cafeterias (5 of 55), fast food establishments (7 of 55), schools (3 of 55), hotels (2 of 55), and restaurants (1 of 55). Sporadic cases were also found at a number of these settings (Table 2). In total, 2,376 stool specimens were collected from both symptomatic and asymptomatic food handlers at different food-catering settings. These specimens were screened by real-time RT-PCR, and norovirus was detected in 449 of 2,376 (19%) specimens. Real-time RT-PCR can distinguish between norovirus GI and GII sequences, and both GI and GII sequences were detected. Twenty-six of 2,376 (1%) stool specimens were positive for norovirus GI sequences, and 423 of 2,376 (18%) were positive for norovirus GII sequences. Noroviruses were detected in specimens from both symptomatic and asymptomatic food handlers (see Tables 1 and 2).

Genotyping and phylogenetic analysis of norovirus. To confirm the positive real-time RT-PCR results and determine the genotypes, we reamplified and sequenced the partial capsid gene. We simplified the phylogenetic trees to include only unique sequences (italicized in Table 1, specimen number column); that is, when two or more sequences from the same outbreak had 100% nucleotide similarity, we named a single consensus sequence, and if a sequence had one or more nucleotide mismatches with others in the same outbreak, we gave the sequence a distinct name. All 26 norovirus GI-positive specimens (from 25 outbreak cases and 1 sporadic case) were sequenced, and these clustered into four distinct genotypes: GI/3, GI/4, GI/8, and GI/14 (Fig. 2). One or more norovirus GII-positive specimens in each setting were confirmed by RT-PCR and sequenced. The GII sequences belonged to 12 genotypes, including one new genotype: GII/1, GII/2, GII/3, GII/4, GII/5, GII/6, GII/7, GII/8, GII/9, GII/10, GII/14, and GII/New (Fig. 3).

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FIG. 2. Phylogenetic tree of the norovirus GI sequences detected in this study (italicized specimen numbers). Norovirus nucleotide sequences were constructed with the partial N-terminal capsid region (14) by using the norovirus Hawaii GII sequence as an outgroup. We simplified the tree to include only unique sequences; that is, when two or more sequences in the same outbreak had 100% nucleotide similarity, we named a single consensus sequence, and if a sequence had one or more nucleotide mismatches with others in the same outbreak, we gave the sequence a distinct name. The numbers on the branches are the bootstrap values for the clusters. Bootstrap values of 950 or higher were considered statistically significant for the grouping (14).

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FIG. 3. Phylogenetic tree of the norovirus GII sequences detected in this study (italicized specimen numbers). Norovirus nucleotide sequences were constructed with the partial N-terminal capsid region (14) by using the Norwalk virus GI sequence as an outgroup.

Molecular epidemiology of outbreaks. Norovirus GI sequences were detected in 5 of 55 (9%) outbreaks (Table 1). We found 14 of 20 (70%) symptomatic food handlers and 11 of 59 (19%) asymptomatic food handlers positive for norovirus GI strains. Sequence analysis showed that each of the five norovirus GI-associated outbreaks was caused by a single norovirus GI strain; that is, two or more sequences from the same outbreak shared 100% nucleotide similarity. Norovirus GII sequences were detected in 50 of 55 (91%) outbreaks. We found 267 of 364 (73%) symptomatic food handlers and 122 of 1,786 (7%) asymptomatic food handlers positive for norovirus GII strains. In 37 of 50 norovirus GII-associated gastroenteritis outbreaks, a single norovirus GII strain was assumed to be responsible, since two or more sequences from the same outbreak shared 100% nucleotide similarity. In the remainder of the GII-associated outbreaks (13 of 50 outbreaks; settings 3, 8, 9, 10, 17, 23, 34, 38, 48, 49, 51, 62, and 68), several norovirus GII sequences were detected; that is, there were one or more nucleotide changes or different genotypes (Table 1). For example, in the norovirus GII-associated setting 48 (Table 2), we detected three norovirus genotypes (GII/3, GII/5, and GII/6) in stool specimens collected from different symptomatic food handlers in the same hotel between 10 and 16 January 2006, whereas for setting 8, a nursing care center, we detected different GII/4 sequences on 25 November 2006 (specimens 401 and 402) (Fig. 3). Interestingly, symptomatic and asymptomatic food handlers were positive by real-time RT-PCR in 9 of these 13 mixed norovirus GII outbreaks (settings 3, 9, 10, 17, 23, 34, 49, 51, and 68). For example, in the norovirus GII-associated setting 34 (Table 1), we detected three norovirus genotypes (GII/6, GII/4, and GII/2); the former two sequences (specimens 52 and 53) were detected in specimens from symptomatic food handlers, and the latter sequence (specimen 237) was detected in a specimen from an asymptomatic food handler.

GII/4 sequences were predominant, accounting for 20 of 55 (36%) outbreaks, excluding the outbreaks with multiple norovirus genotypes (Fig. 4). A considerable number of outbreaks (7 of 55) were also caused by strains belonging to GII/3, followed by GII/5, which caused 3 outbreaks. Interestingly, in 12 of 13 mixed-GII-genotype outbreaks (settings 3, 8, 9, 10, 17, 23, 34, 38, 49, 51, 62, and 68), we always detected a norovirus GII/4 sequence (Table 1).

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FIG. 4. Number of outbreaks caused by each genotype. Only outbreaks due to a single genotype are included. For example, norovirus GII-associated outbreak 8 had two GII/4 sequences and was therefore considered to be a GII/4-associated outbreak, but the GII-associated outbreak 9 had GII/4 and GII/5 sequences, so the cause of the outbreak was unknown, and it was excluded from the figure.

Molecular epidemiology of sporadic cases. Among 35 sporadic cases, norovirus GI sequences were detected in 1 case (3%), which was caused by a GI/4 strain (Table 2). Norovirus GII sequences were detected in 34 of 35 (97%) sporadic cases. The majority of the GII sporadic cases were caused by GII/4 strains (16 of 35), followed by GII/3 (9 of 35). Interestingly, for sporadic cases, only food handlers with symptoms of gastroenteritis were positive for norovirus; 35 of 49 (71%) were positive for GII strains, and 1 of 1 (100%) was positive for GI strains.

Genetic analysis. The partial capsid sequence was used to describe the genetic diversity of the norovirus sequences. All sequences detected in this study closely matched other, published sequences (Fig. 2 and 3). The GII/4 sequences shared >95% nucleotide similarity, although there was noticeable subclustering within the GII/4 genotype (Fig. 3). Norovirus GII/3 also appeared to have subclusters, while the GII/3 sequences shared >96% nucleotide similarity (Fig. 3). Of interest was the detection of a novel GII genotype (GII/New; sequence 299). As shown in Fig. 5, the amino acid start sequence for the capsid was MRM, whereas all other norovirus GII sequences had MKM. To further investigate this finding, we sequenced the entire capsid gene. Only one other full-length capsid sequence (norovirus strain NLVJ23; accession number GenBank AY130762) closely matched the norovirus 299 sequence, with 99.3% amino acid identity over the entire capsid gene. Interestingly, norovirus NLVJ23 also had the unusual amino acid start sequence of MRM for the capsid. The N-terminal regions of the capsids of these two sequences were quite unlike that of any other norovirus GII sequence. In addition, both norovirus 299 and NLVJ23 had an amino acid insertion at the 10th residue, whereas no other norovirus GII sequences had any insertions or deletions within the first 149 amino acids of the capsid gene (Fig. 5).

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FIG. 5. Amino acid alignment of the partial N-terminal-region capsid sequences of the norovirus GII strains. The predicted highly conserved MKM capsid start sequence is boxed. Asterisks indicate conserved amino acids among these capsid sequences.

Analysis of norovirus loads. The real-time RT-PCR results were used to analyze the viral loads by genotype and genogroup (Fig. 6). All positive sequences (not just the consensus sequences shown in the trees) were used for this analysis. The numbers of positive specimens examined for each genotype were as follows: 6 for GI/3, 6 for GI/4, 3 for GI/8, 4 for GI/14, 6 for GII/1, 7 for GII/2, 35 for GII/3, 96 for GII/4, 13 for GII/5, 11 for GII/6, 2 for GII/7, 3 for GII/8, 1 for GII/9, 3 for GII/10, 2 for GII/14, and 1 for GII/new (Fig. 6). Overall, norovirus GII had a slightly higher mean viral load (1 log unit higher) than norovirus GI (3.81 x 10⁸ versus 2.79 x 10⁷ copies/g of stool) (Fig. 6). The highest viral load for norovirus GI was 2.02 x 10⁸ copies/g of stool for GI/4, and the highest viral load for GII was 7.96 x 10⁹ copies/g of stool for GII/4 (data not shown).

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FIG. 6. Viral loads for GI and GII noroviruses. Dotted lines represent the mean for each genogroup.

A comparison between the mean viral loads of symptomatic and asymptomatic individuals found that GI-infected symptomatic individuals had a slightly higher mean viral load than GI-infected asymptomatic individuals (4.43 x 10⁷ versus 3.79 x 10⁶ copies/g of stool), whereas GII-infected symptomatic and asymptomatic individuals had similar mean viral loads (3.31 x 10⁸ versus 5.53 x 10⁸ copies/g of stool). Our results also showed that GI/4-infected symptomatic individuals had a slightly higher mean viral load than GI/4-infected asymptomatic individuals (6.73 x 10⁷ versus 6.34 x 10⁶ copies/g of stool), whereas GII/4-infected symptomatic individuals had a mean viral load similar to that of GII/4-infected asymptomatic individuals (2.17 x 10⁸ versus 6.58 x 10⁸ copies/g of stool). Comparisons between the other genotypes were difficult to perform because the asymptomatic individuals were not infected with those genotypes (data not shown).

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Despite the fact that human noroviruses remain uncultivable, the past decade has witnessed vast improvements in norovirus detection methods, surveillance, and awareness. Methods such as real-time RT-PCR have enabled rapid, broadly reactive, and highly sensitive screening. Numerous molecular epidemiological studies have increased our understanding of these viruses. On the other hand, the number of norovirus infections still remains high, and norovirus infection remains a major health problem worldwide.

In this study we examined 55 outbreaks and 35 sporadic cases of norovirus-associated gastroenteritis in food-catering settings throughout Japan that occurred between 10 November 2005 and 9 December 2006 (Fig. 1). Stool specimens were collected from both symptomatic and asymptomatic food handlers so that the transmission route could be determined. Norovirus was detected in 449 of 2,376 (19%) specimens. Norovirus GI and GII sequences were detected in 9% and 91% of outbreaks, respectively (Tables 1 and 2). Norovirus GI and GII sequences were also detected in 3% and 97% of sporadic cases, respectively. In total, four GI genotypes and 12 GII genotypes, including one new GII genotype (GII/New), were detected during the study period (Fig. 2 and 3).

All of the norovirus GI-associated outbreaks were caused by a single norovirus GI genotype, whereas a single norovirus GII genotype was detected in 38 of 50 norovirus GII-associated outbreaks. In the remainder of the norovirus GII-associated outbreaks (13 of 50 outbreaks), multiple norovirus GII sequences with mismatches or different genotypes were detected (Tables 1 and 2). The GII/4 strains appeared to be the dominant cause of the outbreaks. GII/4 strains were detected in 20 of 55 (36%) outbreaks, followed by GII/3, which was detected in 7 of 55 outbreaks. We also found that noroviruses belonging to GII/4 were the dominant cause of outbreaks in Taiwan (32). In a number of norovirus GII-associated outbreaks (9 of 50 outbreaks), different norovirus genotypes were detected in specimens from symptomatic and asymptomatic food handlers from the same food-catering setting. Interestingly, many of the asymptomatic food handlers were also positive for a norovirus GII/4 sequence, although we cannot be certain whether the subject(s) later developed symptoms (Table 1). Nevertheless, taken as a whole, these results have shown that asymptomatic infections were widespread in the food-catering industry in Japan at the time of the study. Recently, excretion of norovirus by symptomatic and asymptomatic individuals during a hospital outbreak of gastroenteritis where a GII/4 strain was dominant has been described (6). At present, norovirus GII/4 strains are the most prevalent in many countries (4, 8, 27, 28). What is more, variant GII/4 sequences, i.e., those differing by approximately 5% of amino acids, were speculated to be more virulent and part of the reason for the increased number of infections worldwide (8). Clearly, norovirus GII/4 strains are widespread, although they may not always cause symptoms, which may account for the increased number of infections via a "silent" (that is, asymptomatic) transmission route. GII/4 strains were also the dominant cause of the sporadic cases; they were detected in 16 of 35 (46%) sporadic cases. This result suggests that the GII/4 strains are an important cause of both outbreaks and sporadic occurrences of gastroenteritis.

Noroviruses can be transmitted by the fecal-oral route through person-to-person contact and by food- and waterborne infections (2, 3, 12). In Japan, oyster-associated gastroenteritis is a major problem, and it is not unusual to detect multiple norovirus genotypes in an oyster-associated outbreak (14). In this study, multiple norovirus GII sequences were detected in the same outbreak, but norovirus GI and GII sequences were not detected in the same outbreak. The low infectious dose (22) and prolonged shedding (11) of norovirus makes transmission almost certain, although we could not be certain whether foods were contaminated. Mean viral loads of GI and GII were found to be 2.79 x 10⁷ and 3.81 x 10⁸ copies/g of stool, respectively (Fig. 6). Similar viral loads were found for infected symptomatic and asymptomatic individuals, indicating the potential hazard of these highly contagious viruses. In a recent study in which the number of norovirus cDNA copies per gram of stool specimen was analyzed, a discrepancy was found between the different norovirus genogroups. Chan et al. found median viral loads of 8.4 x 10⁵ and 3.0 x 10⁸ copies/g of stool specimen for norovirus GI and GII, respectively, and speculated that the higher viral loads of GII strains were due to their higher transmissibility (5). Of note, our results showed that GII/4 strains had the highest mean viral load overall (7.96 x 10⁹ copies/g of stool), further increasing the clinical importance of this dominating genotype.

Norovirus GII capsid sequences are highly conserved at the N-terminal region and, to the best of our knowledge, share an identical amino acid start sequence, MKM. In this study, we identified an atypical norovirus GII capsid amino acid start sequence, MRM, in the norovirus 299 sequence (Fig. 5). What is more, the MRM amino acid residues did not match other norovirus genogroups, and only one other closely matching sequence was found in the database (norovirus strain NLVJ23). Interestingly, the N-terminal capsid region is highly conserved in all norovirus GI sequences and shares the same amino acid start sequence, MMM. This suggests that either norovirus 299 and NLVJ23 may not belong to norovirus GII or the N-terminal capsid region is not as highly conserved in each genogroup as previously anticipated.

In conclusion, we found that norovirus infections were a common cause of gastroenteritis in the food-catering industry in Japan. Our results have also shown that asymptomatic infections with noroviruses, whether with a sequence identical to that infecting a symptomatic food handler or with a distinct sequence, were widespread in the food-catering industry. Much work is needed to curb the burden of this disease and reduce its transmission. A simple workplace policy that will protect ill workers and allow for paid leave may not be sufficient to stop transmission, since asymptomatic food handlers may continue to work.

 
This work was supported in part by a grant for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labor and Welfare of Japan and by a grant for Research on Health Science Focusing on Drug Innovation from The Japan Health Science Foundation.

 
* Corresponding author. Mailing address: Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-murayama, Tokyo 208-0011, Japan. Phone: 81-42-561-0771. Fax: 81-42-561-4729. E-mail: ghansman{at}nih.go.jp

Published ahead of print on 10 October 2007.

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Journal of Clinical Microbiology, December 2007, p. 3996-4005, Vol. 45, No. 12
0095-1137/07/$08.00+0     doi:10.1128/JCM.01516-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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