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ISSN : 1229-1153(Print)
ISSN : 2465-9223(Online)
Journal of Food Hygiene and Safety Vol.32 No.6 pp.455-459
DOI : https://doi.org/10.13103/JFHS.2017.32.6.455

Molecular Epidemiology of Norovirus in Asymptomatic Food Handlers in South Korea

Jeong Su Lee, Min Hee Jeong, Si Yeon Ju, Kyung Ah Kang, In Sun Joo*
Food Microbiology Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Osong, Korea
Correspondence to: In Sun Joo, Food Microbiology Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Osong 28159, Korea 82-43-719-4302,82-43-719-4300jis901@korea.kr
20170428 20170520 20171011

Abstract

Norovirus (NoV) is the most common cause of acute gastroenteritis in all age groups worldwide. In this study, prevalence of asymptomatic norovirus infection was investigated in food handler being employed at food catering facilities in South Korea. A total of 2,729 fecal specimens from asymptomatic food handlers were analyzed, and 1.06% of food handlers (29/2,729) had asymptomatic NoV infection. Of these, 17.2% (5/29) were positive for NoV GI and 82.7% (24/29) were positive for NoV GII. Especially, sequencing and phylogenetic analysis showed that GII-4 was the most prevalent genotype and a large number of asymptomatic food handlers were infested with norovirus GII-4 strains. The results of this study show that asymptomatic food handlers may be potential transmission sources for NoV infection. These results emphasize the need for training of food catering employees about norovirus prevention. Asymptomatic norovirus infection should receive more attention.


초록


    Ministry of Food and Drug Safety
    121612KFDA033

    The positive-sense polyadenylated single-stranded RNA virus family Caliciviridae contains four genera: Vesivirus, Sapovirus, Lagovirus, and Norovirus (NoV)1). The NoV is currently classified into 6 genogroups (GI to GVI)2), and only NoV GI, GII, and GIV have been associated with human gastroenteritis3). The GI and GII genogroups of human NoV are further classified into 9 and 22 genotypes, respectively4). The virus is transmitted predominantly through ingestion of contaminated food as well as person-to-person by the fecal-oral route, airborne transmission and contact with contaminated surfaces5). In previous reports, infected food handlers have been implicated repeatedly as the source of infection in several outbreaks6,7). Asymptomatic NoV infections of food handlers may play a role in transmission8). NoV disease outbreaks are reported year-round in Korea. Furthermore, there have been several large outbreaks of NoV since 20039,10). The NoV outbreak has exhibited a high prevalence during winter with cold temperate climates10). In other words, the environment with low temperature (average 2.1°C) and humidity (average 59.6%) may be a possible contributors to the transmission of enteric infections12). Many studies have reported the monitoring of NoV in facilities with outbreak11,13,14). However, little research is available about circulating viral strains in asymptomatic individuals in facilities without NoV outbreaks. Recently, another epidemiologic study of NoV outbreak in South Korea has reported that the excretion of NoV from asymptomatic food handlers may be an important portion of NoV outbreak 9,12,14,17). The aims of this study were to investigate the molecular epidemiological characteristics of NoV detection from asymptomatic food handlers working at food catering facilities in South Korea from October 2012 to April 2013.

    Materials and Methods

    Clinical samples

    Rectal swab samples were collected from asymptomatic food handlers during regular physical examinations at five health centers (Tongyeong, Gyeonggi, Yeosu, Taean and Chenogju) in South Korea. Among 2,729 food handlers, the gender ratio of this study was 72% (1,965) female and 28% (764) male. Swab samples collected from randomly selected food handlers distributed in South Korea were suspended in 2 mL of phosphate buffered saline (pH 7.2), vortexed slightly, and then centrifugated at 3,000 rpm for 10 min to separate the supernatants. Supernatants were stored at −80°C until use.

    Viral RNA extraction

    The QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) was used to perform the viral RNA extraction in accordance with manufacturer's instructions. Viral RNA was eluted in 60 μL of AVE buffer and stored at −80°C until use.

    Conventional nested RT-PCR

    NoV was identified using conventional nested RT-PCR as previously described17). PCR amplification was performed to detect the ORF 1-2 junction region of the NoV. 5 μL of extracted RNA was used in the RT-PCR mixture with a total volume of 25 μL comprised of 10 μL one-step RT-PCR premix (with AMV reverse transcriptase), 6 μL of distilled water and 2 μL of each NoV GI and GII primer (20 pmol) (Table 1). The cycling conditions were: 30 min at 45°C for cDNA synthesis, 5 min at 94°C for predenaturation, then 35 cycles consisting of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min 30 sec. The semi-nested PCR amplification procedure was followed using the first-round amplicon. 2 μL of amplicon was added to 48 μL of the PCR mixture containing 5 μL of 10× reaction buffer (Bioneer, Daejeon, Korea), 4 μL 10 mM dNTPs (Bioneer, Daejeon, Korea), 2.5 μL of each NoV GI and GII primer (20 pM), 1 μL of Top DNA polymerase (Bioneer, Daejeon, Korea) and 33 μL distilled water. The cycling conditions were: 5 min at 94°C for predenaturation, then 25 cycles consisting of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min 30 sec. The amplification products were analyzed by 2% agarose gel electrophoresis and visualized with ultraviolet (UV) light after ethidium bromide staining. Samples that were NoV positive by conventional RT-PCR were further characterized (genotyped) by DNA sequencing. All PCR products were sequenced using an ABI Prism 3500×L genetic analyzer and BigDye Terminator cycle sequencing mix (Applied Biosystems, Foster City, CA, USA). For genotyping of sequenced products, the sequences were compared to those in the GenBank database using the NCBI BLAST search program. To confirm the genotype of NoV, phylogenetic analysis was performed and the phylogenetic trees were obtained using the CLUSTAL W method and MegAlign (Lasegene, DNAstar, Inc. Madison, WI, USA) software.

    Real time RT-PCR

    To analyze viral copy number within each sample, NoVs were amplified with a one-step real time RT-PCR kit (Ambion, Austin, TX, USA) as previously described17). The real time RT-PCR reaction mixture contained 5 μL of extracted RNA, 12 μL of 2× RT-PCR buffer (Ambion, Austin, TX, USA), 1 μL of each NoV GI and GII primer (10 pM), 0.5 μL of each fluorescent probe (10 pM), 0.5 μL of 25× enzyme mix, 1.5 μL of enhancer, and 3 μL of distilled water. 5 μL of extracted NoV sample was added to each well, and the final total volume was 25 μL (Table 2). The cycling conditions were: reverse transcription at 45°C for 30 min, predenaturation at 95°C for 10 min, and 45 cycles of denaturation at 95°C for 15 sec and annealing, and extension at 56°C for 1 min.

    Results and Discussion

    Among 2,729 food handlers comprised of 764 male and 1,965 female, asymptomatic infection was detected in 29 (1.06%) by conventional nested RT-PCR. The prevalence of NoV in asymptomatic food handlers was 58.62% during winter season (from December to February). Especially, there was a tendency that the prevalence of asymptomatic NoV infection in January has higher detection than other months (Table 3). Among 29 samples, 5 GI-positive samples and 24 GII-positive samples were identified by real time RTPCR. Mean viral load in stool specimens for GI NoV was 6.1 × 107 viruses/g (range, 2.1 × 102 to 3.0 × 108 viruses/g) and was 6.1 × 104 viruses/g (range, 1.5 × 101 to 9.7 × 105 viruses/g) for GII (data not shown). Sequencing and phylogenetic analysis showed that the 5 GI-positive samples were genotyped as GI-4, GI-6, GI-7, GI-9 and 24 GIIpositive samples were GII-2, GII-3, GII-4, GII-6, GII-16, GII-17. Our data demonstrated that mean viral load in stool specimens of GI NoV was higher than that of GII NoV. The genotypic distribution of the 29 NoV strains was as follows: GI-4, 6.89% (n=2); GI-6, 3.44% (n=1); GI-7, 3.44% (n=1); GI-9, 3.44% (n=1); GII-4, 41.37% (n=12); GII-17, 20.69% (n=6); GII-2, 10.34% (n=3); GII-3, 3.44% (n=1); GII-6, 3.44% (n=1); GII-16, 3.44% (n=1) (Table 4).

    Molecular epidemiological studies of NoV strains in asymptomatic food handlers have reported that GII-4 was dominant in transmissibility1,16,18). Likewise, in our study, the NoV GII-4 strain was more prevalent than others. This is similar to the previous Japanese study investigating NoV outbreaks, in which the NoV samples were collected from asymptomatic food handlers in a hotel and confirmed belonging to NoV GII2,4). Accordingly, it is possible to assume that GII is the dominant genogroup in an asymptomatic food handler population. This might be related to the fact that genogroup II strains (especially GII-4) are more transmissible than the others4,9). The clinical manifestations (e.g., increased vomiting) of GII-4 strain infections or physical characteristics (e.g., environmental persistence) of this strain might facilitate spreading. In South Korea, previous study that the vehicle of transmission in this outbreak was dried radish salad prepared by the food handler and served to students in the elementary school22). Furthermore, approximately 50% of all NoV outbreaks in the United States are linked to ill food-handlers20). In this regard, asymptomatic carriers of Nov are main cause related to the occurrence of NoV outbreaks 9,15,21).

    In this study, phylogenetic analysis was used to evaluate the relatedness of NoV strains detected and to compare their partial capsid sequence with those of GI and GII genogroup reference strains (Fig. 1). In phylogenetic analysis with GI genogroup, FH-R4-KR8 (KF773988) and FH-R5-KR1 (KF- 773980) strains were identified as genotype GI-4. 3 strains, FH-R1-KR5 (KF773995), FH-R1-KR10 (KF774000) and FH-R4-KR12 (KF774002) were analyzed as GI-6, GI-7 and GI-9, respectively. The FH-R4-KR8 (KF773988) and FHR5- KR1 (KF773980) strains were clustered into the GI-4 NV34 (KF049146) with 97.0% and 89.9% identity, respectively. Sequence analysis revealed that strain FH-R1- KR5 (KF773995) shared the greatest identity with strain GI- 6 BS-5 (AF093797) (92.5%). Strains FH-R1-KR10 (KF- 774000) and FH-R4-KR12 (KF774002) were found to be related most closely to GI-7 Miyagi-JP (AB758449) (90.3%) and GI-9 Vancouver730 (HQ637267) (94.5%), respectively. The NoV GII strains were 87.3 to 97.6% homologous with the reference GII-4, 89.9 to 92.1% with the reference GII- 17, 90.0 to 96.7% with the reference GII-2, 91.6% with the reference GII-3, 93.5% with the reference GII-6 and 95.3% with the reference GII-16. FH-R4-KR3 (KF773983), FHR4- KR6 (KF773986), FH-R4-KR5 (KF773985), FH-R3- KR4 (KF773978), FH-R4-KR4 (KF773984), FH-R1-KR7 (KF773997), FH-R1-KR8 (KF773998), FH-R1-KR3 (KF7- 73993), FH-R1-KR4 (KF773994), FH-R3-KR5 (KF773979), FH-R3-KR1 (KF773975), and FH-R4-KR2 (KF773982) stains showed 87.3% to 97.6% sequence identity to the GII- 4 genotype, suggesting that GII-4 is the most prevalent genotype in an asymptomatic Nov infection.

    The results of this study showed that asymptomatic employees as well as symptomatic food handlers may contribute infection as a potential transmission source in NoV outbreaks. To reduce food contamination, strict general hygiene practices should be implemented. More attention should be paid to facilities for food workers to reinforce hand hygiene practices and prevent foodborne outbreak.

    Nucleotide sequence accession numbers

    The nucleotide sequence data have been submitted to GenBank and assigned accession numbers KF773975 to KF774002.

    Acknowledgements

    We thank the staffs at 5 centers for collecting stool specimens of food handler. This study was supported by a grant (121612KFDA033) from Ministry of Food and Drug Safety in 2013.

    Figure

    JFHS-32-455_F1.gif

    Phylogenetic tree of NoV detected in asymptomatic food handler. Neighbor-joining phylogenetic tree based on nucleotide sequences of the capsid region of the NoV genome (A, norovirus GI; B, norovirus GII). The numbers in the branches indicate the bootstrap values. Reference strains of NoV selected from Genbank are indicated by accession numbers. The scaled indicates nucleotide substitutions per position.

    Table

    Primers used for NoV detection by conventional PCR

    Primers and probes used for NoV detection by real-time RT-PCR

    The number of detection (per months)

    The genotypic distribution of detection strains

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