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Norovirus contamination in retail oysters from Beijing and Qingdao, China
Food Control 86 (2018) 415e419
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Norovirus contamination in retail oysters from Beijing and Qingdao, China amus Fanning a, b, c, Li Nan a, Wang Jiahui a, Jiang Tao a, Han Chunhui a, Se Zhang Hongyuan a, Zhang Jing a, Li Fengqin a, * a
Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing, China UCD-Centre for Food Safety, School of Public Health, Physiotherapy and Sports Science, University College Dublin, Belfield, Dublin D04 N2E5, Ireland Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, United Kingdom b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 August 2017 Received in revised form 27 November 2017 Accepted 30 November 2017 Available online 5 December 2017
The consumption of raw oyster has been linked to numerous foodborne gastroenteritis outbreaks caused by norovirus (NoV) in different countries of the world, including in China. This study investigated the prevalence of NoV and contamination levels in retail oysters taken from markets in Beijing and Qingdao in China. The oysters were collected monthly from seafood markets between September 2015 and September 2016 (13 months) in both cities. The digestive glands of these oysters were dissected and NoV particles were extracted. Viral RNA was detected using a TaqMan-based real time one-step reverse transcription-PCR (qRT-PCR) protocol. Of total 672 oyster samples assessed, 652 were considered to be valid for inclusion in the study. The prevalence of NoV was 20.71% (135/652), and this included 21.48% (29/135) of the samples that were positive for the GI strain alone, 62.96% (85/135) contained the GII strain alone and 15.56% (21/135) were mixed with GI and GII, respectively. A total of 68 NoV-positive samples were quantified by qRT-PCR and values obtained ranged from 3.55 103 to 1.45 106 genomic copies per g digestive tissue. The NoV contamination in retail oysters fluctuated with the sampling month and peaks of contamination occurred in February (49.12%) and March (55.36%) 2016, respectively. Considering some of the oysters were consumed as raw or half-cooked in China, as a risk reduction measure these oysters should be well heated prior to consumption. Furthermore, successive and region-extended monitoring in retail oysters for NoV as well as risk communication is recommended. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Norovirus oyster contamination realtime reverse transcription-polymerase chain reaction (qRT-PCR)
1. Introduction Norovirus (NoV) is known as the leading cause of human nonbacterial acute gastroenteritis that can infect individuals of all age groups worldwide (Hall, 2012). NoV is a non-enveloped virus with a single-stranded, positive-sense RNA genome. It is belonging to genus Norovirus, family Caliciviridae (Kroneman et al., 2013). NoV genus is currently divided into 6 distinct genogroups (denoted as GI through to GVI), each of which has been subdivided into multiple genotypes (Zheng et al., 2006; Kroneman et al., 2013). GI and GII, can be subdivided into 9 and 22 genotypes respectively,
* Corresponding author. No.7, Panjiayuan Nanli, Chaoyang District, Beijing, 100021, PR China. E-mail address: [email protected] (L. Fengqin). https://doi.org/10.1016/j.foodcont.2017.11.043 0956-7135/© 2017 Elsevier Ltd. All rights reserved.
and both of these constitute the primary strains epidemiologically linked to human cases of acute gastroenteritis (Liao et al., 2016). Norovirus infections constitute a serious disease burden both in developed and developing countries. According to the U.S. Centers for Disease Control and Prevention (US CDC), NoV was responsible for 60% of acute gastroenteritis cases or 21 million cases in the United States each year (Patel et al., 2009). Children, the elderly and immunocompromised persons may experience more severe symptoms and/or extended duration of illness or even chronic diarrhea. There are an estimated 70,000 norovirus-associated deaths recorded among children <5 years annually worldwide (Lanata et al., 2013). In China, reports of acute gastroenteritis cases caused by NoV have been increasing and these now account for 60e96% of nonbacterial infectious diarrhea recorded since 2012 (Liao et al., 2016). Norovirus is mainly transmitted via the fecal-oral route and
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contaminated food is one of the important vehicles for transmission. According to US CDC, of 348 NoV outbreaks reported from January 1996 to November 2000, the sources included food (39%), person-to-person contact (20%) and water (3%) (Thornton, Jennings-Conklin, & McCormick, 2004). Shellfish, especially oysters, are a recognized transmission vector for human NoV in the environment (Yu et al., 2015). These shellfish can filter large volumes of water as part of their filter-feeding activities and consequently accumulate and concentrate the virus in their digestive gland. Furthermore, because oysters are typically grown in coastal water that potentially is contaminated by human waste along with the fact that oysters are often consumed under-cooked or raw, this food matrix presents a high risk for viral infections, frequently being involved in NoV outbreaks (Webby et al., 2007). On the basis of data provided by European Food Alert System for Food and Feed (RASFF), all but 3 of the 36 outbreak notifications involving viruses reported during an 11-year period (2000e2011) were attributed to NoV. Among these, 22 were associated with oysters (Le Guyader, Atmar, & Le Pendu, 2012). In addition, secondary transmission from person-to-person may occur, and outbreaks may give rise to school and workplace closures, as well as the closure of oyster harvesting waters along with recalls. Although the adoption of regulations that specify acceptable levels of bacterial enteric pathogens in shellfish tissues or in shellfish-growing water has significantly decreased the impact of bacteria as causes of shellfishassociated disease outbreaks, these regulations have failed to prevent many outbreaks of viral origin (Le Guyader et al., 2009). China is one of the largest countries in the world for consumption and production of oysters. Little information is available to describe oyster contamination with NoV. To protect consumer health in China, the NoV contamination in retail oysters sourced in Beijing and Qingdao cities were monitored over a 13-month period. Data obtained reported on the quantification of this viral genomic copies in the retail oysters collected. Further the temporal geographical distribution of oyster-related NoV was described in order to extend our knowledge of NoV prevalence in China. 2. Materials and methods 2.1. Oyster sampling A total of 672 fresh oyster samples were randomly collected from one seafood wholesale market in Beijing and in addition six retail markets in Qingdao from September 2015 to September 2016 (covering a period of 13 months). This accounted for approximately 30e40 samples per month. The seafood wholesale market recruited to the study was the biggest establishment in Beijing and oysters from here were placed routinely for sale in many supermarkets or farmer's markets, being dispatched from this location. On each sampling day, no more than 4 retail outlets were selected for study. A maximum of 30 individual oysters were selected randomly for testing from each of these sampling sites. Samples were transported on ice directly to the laboratory and NoV extraction was carried out within 24 h. 2.2. Oyster sample processing Since the weight of the oysters differed depending on the individual oyster or collection season, each sample was adjusted so that it contained 2e5 individual oysters and make sure each sample contains 2 g digestive gland of oyster. The extraction procedure of NoV from digestive gland of oyster was referred to Microbiology of the food chain–Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR (15216-1), but has some changes in number of oysters as one sample. Briefly the shell of
each oyster was carefully washed with tap-water and the digestive glands dissected carefully from other tissues. All digestive glands for these 2e5 oyster samples were pooled and homogenized thoroughly. A portion of 2 g of homogenized digestive tissues was then transferred into a 50-mL centrifuge tube followed by the addition of 10 mL mengovirus VMC0 (CeeramTooLs, France) as the process control and 2 mL proteinase K (Sigma, U.S.A.) solution with a final concentration of 0.1 mg/mL. This mixture was incubated in a shaking incubator at 280 rpm for 60 min at 37 C and then for a further 15 min in a water bath at 60 C. After centrifugation at 3000 rpm for 5 min at room temperature, the supernatant (approximately 3.0 mL) was collected into a sterile Eppendorf tube and stored at 80 C until required. 2.3. NoV nucleic acid purification Viral RNA was extracted and purified using a QIAamp Viral RNA Mini kit (QIAgen, Hilden, Germany) in a final volume of 50 mL, in accordance with the manufacturer's instructions. The extracted RNA were then used as the template for real time reverse transcription polymerase chain reaction (qRT-PCR) and maintained at 80 C prior to analysis for not longer than one week. 2.4. Real-time RT-PCR amplification Oligonucleotide primers and detection probes employed in this study to identify GI, GII and along with the mengovirus were shown in Table 1. The real-time RT-PCR was carried out using RNA Ultrasense One-step Quantitative RT-PCR System (Invitrogen, U.S.A.). All samples extracted were screened for NoV GI, GII and mengovirus by triplex qRT-PCR, performed in a CFX96 (BIO-RAD,U.S.A.) real-time PCR platform. The GI probe was labelled with 5-hexachloro-fluorescein (HEX) at 50 -end and black hole quenching1(BHQ1) at 30 -end and be detected at 556 nm fluorescence channel. The GII probe was labelled with 6-carboxyfluorescein (FAM) at 50 -end and black hole quenching1(BHQ1) at 30 -end and be detected at 515 nm fluorescence channel. The mengovirus probe was labelled with 5-Nhydroxysuccinimide ester (CY5) at 50 -end and black hole quenching2(BHQ2) at 30 -end and be detected at 667 nm fluorescence channel. The three different gene fragments can be detected simultaneously through different fluorescence channel in each well. The final reaction volume of 20 mL (without nucleic acid template) consisted of 500 nM of each upstream primer; 900 nM of each downstream primer; 250 nM of each detection probe, along with buffer and enzymes at concentrations recommended by the manufacturer. 5 mL of purified nucleic acid or control was added per well, and the final volume in each well was 25 mL. The thermal profile comprised 55 C for 30 min and 95 Cfor 5 min, followed by 45 cycles of 95 C for 15s, 60 C for 1min and 65 C for 1 min. Additionally, highly purified water was used as the blank qRT-PCR control. Phosphate-buffered saline (0.01 mol/L, pH7.2) was used as the negative process control and was run through all stages of the analytical process. The cycle threshold (Ct) was set manually at 0.1, and it was always on the logarithmic portion of the amplification curve thereby being distinguishable from the background fluorescence. Those samples for which reactions yielded a Ct value below or equal to 35 for NoV GI or GII were considered as positive, and the Ct value above or equal to 38 for NoV GI or GII were considered as negative, and the Ct value between 35 and 38, qRT-PCR detection should be repeated. 2.5. Extraction efficiency To control for false-negative signals due to extraction failure, a
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Table 1 Primers and Probes for NoV detection used in the present study. Name GI
GII
Mengo virus
QNIF4(FW) NV1LCR (REV) NVGG1P(PROBE) QNIF(FW) COG2R (REV) QNIFs(PROBE) Mengo110(FW) Mengo209 (REV) Mengo147(PROBE)
Primers and Probes
reference
50 -CGC TGG ATG CGN TTC CAT-30 50 -CCT TAG ACG CCA TCA TCA TTT AC-30 50 -HEX-TGG ACA GGA GAY CGC RAT CT-BQ1-30 50 -ATG TTC AGR TGG ATG AGR TTC TCW GA-30 50 -TCG ACG CCA TCT TCA TTC ACA-30 50 -FAM-AGC ACG TGG GAG GGC GAT CG-BQ1-30 50 -GCG GGT CCT GCC GAA AGT-30 50 -GAA GTA ACA TAT AGA CAG ACG CAC AC-30 50 -CY5-ATCACA TTA CTG GCC GAA GC-BQ2-30
da Silva et al., 2007 Svraka et al., 2007 Svraka et al., 2007 Loisy et al., 2005 Kageyama et al., 2003 Loisy et al., 2005 Pinto, Costafreda, & Bosch, 2009 Pinto et al., 2009 Pinto et al., 2009
process control (mengovirus) was included in each sample. The extraction efficiency of the spiked mengovirus from an individual sample was calculated using the following equation: extraction efficiency (%)¼(the number of recovered mengovirus genomic copies/the number of seeded mengovirus genomic copies) 100%. Samples with the Ct value of GI or GII NoV above or equal to 38 and the extraction efficiency below 1% should be discarded.
2.6. Sensitivity and standard curves of GI and GII NoV The sensitivity and standard curves of GI and GII NoV were determined using a Norovirus GI Q and Norovirus GII Q Standard kit (CeeramTooLs, France) with real-time RT-PCR. A standard curve of GI or GII was generated using a 10-fold series of dilutions ranging from 104 to 101 viral genomic copies per microliter and a 2-fold series of dilutions ranging from 10 to 1.25 viral genomic copies per microliter. Viral genomic copy numbers contained in each of the analyzed samples were determined by comparison with the standard curve. The Norovirus GI Q Standard and Norovirus GII Q Standard were also used as positive control.
3. Results 3.1. Sensitivity and standard curves of GI and GII NoV The sensitivity of the method is 2.5 viral genomic copies per microliter (or 12.5 viral genomic copies per reaction) for GI and GII, and both of them corresponding Ct values were below 38. The limit of quantification was 10 genomic copies per microliter (or 50 viral genomic copies per reaction) for GI and GII. The standard curves of GI and GII were shown in Fig. 1.
3.2. NoV prevalence in retail oysters Among 672 oyster samples analyzed, 20 were determined to have a Ct value above 38 for both GI and GII NoV and thus were excluded as their extraction efficiency was unacceptable (less than 1%). Data obtained from 652 samples were considered to be valid. A total of 135 (20.70%, 135/652) samples were contaminated with NoV, 55 positives were recorded from Beijing and 80 from Qingdao, respectively. Genogrouping data for these 135 NoV-positive samples revealed that 29 (21.48%, 29/135) samples were identified as being contaminated with GI NoV alone, 85 (62.96%, 85/135) by GII NoV alone and 21 (15.56%, 21/135) were mixtures of GI and GII NoV. The prevalence of NoV in retail oysters from different regions was shown in Table 2. In terms of NoV contamination in oysters from Beijing, 55 (16.03%, 55/343) samples were positive for NoV, comprising 12 (21.82%, 12/55) GI strains alone, 37 (67.27%, 37/55) GII strains alone and 6 (10.91%, 6/55) co-contaminated with both GI and GII strains. Comparatively, samples from Qingdao, a coastal city, were more heavily contaminated with norovirus (80/309, 25.89%) than those from Beijing (p ¼ 0.002). The prevalence of NoV genogroups identified in samples taken from Qingdao was 21.25% (17/80) for GI alone, 60.00% (48/80) for GII alone, and 18.75% (15/ 80) for both, respectively. No significant difference in prevalence of any genogroups of NoV detected from oysters between the two cities was observed. 3.3. NoV seasonality The prevalence of NoV in retail oysters varied with the sampling seasons. Contamination appeared to peak in February (49.12%) and March (55.36%); in 2016. A peak of oyster contamination by NoV in the cool and dry months of the year was observed in Beijing (February, 42.22%) and Qingdao (March, 78.12%), respectively. As Qingdao is a coastal city with a large oyster production industry together with high levels of consumption, NoV contamination were also frequently detected in the late autumn with 35.29% of recorded positives occurring in October and 46.88% for November, respectively. No NoV was detected in oysters collected in August 2016 in both cities. Table 3 showed the oyster contamination with NoV as it varied with sampling time. 3.4. Quantification of NoV in naturally contaminated oysters
Fig. 1. Standard curves for real-time RT-PCR detection of GI and GII, respectively.
The number of viral genomic copies in oysters was calculated by multiplying the value generated from the real-time RT-PCR by the dilution factors from the viral and RNA extraction. Accordingly, the limit of quantification (LOQ) was 3.33 103 genomic copies per gram of digestive tissues (DT) for NoV GI and GII. Only those samples wherein the extraction efficiency was equal to or higher than 10% were assigned to perform a quantitative analysis for NoV. A total of 68 samples were therefore available for NoV
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Table 2 Norovirus prevalence in retail oysters from Beijing and Qingdao. Region
Beijing Qingdao Total
No. of samples
343 309 652
No. of positive samples (%)
55 (16.03%) 80 (25.89%) 135 (20.71%)
NoV genogroup No. of GI positive samples (%)
No. of GII positive samples (%)
No. of GI & GII positive samples (%)
12 (21.82%) 17 (21.25%) 29 (21.48%)
37 (67.27%) 48 (60.00%) 85 (62.96%)
6 (10.91%) 15 (18.75%) 21 (15.56%)
Table 3 Comparison of NoV contamination in retail oysters in different months. Month
Sep 2015 Oct 2015 Nov 2015 Dec 2015 Jan 2016 Feb 2016 Mar 2016 Apr 2016 May 2016 Jun 2016 Jul 2016 Aug 2016 Sep 2016
Total Samples
43 59 54 47 34 57 56 77 49 42 42 43 49
Positive Samples (Rate)
4 (9.30%) 12 (20.34%) 18 (33.33%) 2 (4.26%) 4 (11.76%) 28 (49.12%) 31 (55.36%) 6 (7.79%) 9 (18.37%) 8 (19.05%) 2 (4.76%) 0 (0.00%) 11 (22.45%)
Beijing
Qingdao
Total Samples
Positive Samples (Rate)
Total Samples
Positive Samples (Rate)
19 25 22 16 23 45 24 52 24 18 24 27 24
4 (21.05%) 0 (0.00%) 3 (13.64%) 2 (12.50%) 2 (8.70%) 19 (42.22%) 6 (25.00%) 6 (11.54%) 0 (0.00%) 6 (33.33%) 0 (0.00%) 0 (0.00%) 7 (29.17%)
24 34 32 31 11 12 32 25 25 24 18 16 25
0 (0.00%) 12 (35.29%) 15 (46.88%) 0 (0.00%) 2 (18.18%) 9 (75.00%) 25 (78.12%) 0 (0.00%) 9 (36.00%) 2 (8.33%) 2 (11.11%) 0 (0.00%) 4 (16.00%)
quantification. The numbers of GI NoV in these oysters ranged from 3.55 103 to 1.70 104copies/g DT (geometric mean: 4.98 103 copies/g DT), and GII NoV between 5.04 103 and 1.45 106copies/ g DT (geometric mean: 7.04 104 copies/g DT) (Table 4). The highest quantities of NoV copies for GI and GII in oysters were found in April 2016 and October 2015, respectively.
4. Discussion Shellfish production is one of the most important economic activities in coastal areas of China. Most oysters produced are consumed as raw or half-cooked and this feature of consumption presents a high risk for viral infections, in humans, often leading to NoV outbreaks (Yu et al., 2015). This study was focused on the prevalence and contamination levels of detectable NoV in oysters, available for purchase in major population centers in China. For NoV detection, a sensitive and reproducible method was required. At present, the methods for NoV detection include electron microscopy (or immune electron microscopy), enzyme immune assay and molecular methods. qRT-PCR with high sensitivity and specificity was considered to be a useful method for the detection of NoV in foods (Pan, 2015). In this study a tailored qRTPCR method for NoV detection was developed and used in the survey of NoV in oysters. As NoV was difficult to culture in vitro and we cannot get enough NoV particles with definite number to spike sample, so the limit of detection (LOD) and the limit of
quantification (LOQ) were more theoretical LOD and LOQ. The result maybe overestimate the sensitivity of the method and underestimated the content of NoV genomic copies in samples. Beijing is an international metropolis. Foods produced all over the country and many part of the world can be consumed in Beijing. In contrast, Qingdao is a coastal city in northern China and oyster farming is one of the important economic activities. Both of these cities were considered as useful sampling pools for oysters potentially contaminated with NoV. The results of this study showed that the average prevalence of NoV in retail oysters was 20.71% a figure that fluctuates with the sampling season. The contamination rate in winter was higher than that in summer. It was reported previously that oyster glycogen played an important role in NoV binding and it is present at high levels in the flesh of shellfish from November through March (Furuya et al., 2005; Le Guyader et al., 2006; Burkhardt and Calci, 2000), a period coinciding with seasonal outbreaks of NoV. The prevalence of NoV GII(62.96%) was higher than that of GI(21.48%) in the retail oysters and only 21 samples (15.56%) were co-infected by GI and GII. This result differed from observations published earlier wherein it was reported that GI NoV was detected more often than GII in shellfish. (Maalouf et al., 2011; Li et al., 2014). Previously it was suggested that the probability of NoV infection was dose dependent (Bok et al., 2009) and the infectious dose was suggested to be about 18-2800 infectious virus particles (Teunis et al., 2008; Atmar et al., 2014). In this study, the number of NoV
Table 4 Quantification of NoV contamination in oysters. NoV genotype
No. of samples
Geometric concentration of GI NoV (copies/g DT) maximum
GI GII GIþGII
15 51 2
4
1.70 10 e 6.80 103
minimum 3
3.55 10 e 3.79 103
Geometric concentration of GII NoV (copies/g DT) geometric mean 3
4.98 10 e 5.08 103
maximum
minimum
geometric mean
e 1.45 106 1.28 104
e 5.04 103 8.83 103
e 7.04 104 1.06 104
J. Tao et al. / Food Control 86 (2018) 415e419
was determined by genomic copies. The data in this study may overestimate the risk of oyster contamination and human infections, as the latter strategy does not relate to the infectious dose. Further, quantification of NoV is regarded as challenging as it may produce considerable variation in results generated by different researchers because of different PCR parameters, reagents, NoV standards (e.g., double-stranded DNA plasmids or synthesized RNA) and even different machines. Therefore, NoV prevalence data and the detection methods used in the specific study must be considered when evaluating results. In summary, a high prevalence of NoV contamination was observed in oysters placed on the retail market for sale and the contamination levels of NoV may result in gastroenteritis symptoms in consumers. Considering some oysters were consumed as raw or lightly-cooked, from a public health view point, it suggests that as a risk reduction measure, consumers should be encouraged to cook oysters. Results of this study will provide the basic information required for risk assessment on dietary exposure to NoV via oysters, and these data will assist policy-makers to develop strategies to limit NoV contamination of oysters and its transmission to humans. Surveillance of NoV contamination in marketed oysters is highly recommended, as a means of extending the epidemiological data available to describe this risk to human health. Additionally, public education and risk communication is the key to prevent NoV gastroenteritis, in the future. Funding This work was supported by the Beijing Natural Science Foundation (grant number: 5141002). Conflicts of interest All authors listed in the manuscript contributed to conception, acquisition, analysis and interpretation of data, design of the manuscript, critically revised the manuscript and approved the final submitted version. The authors have no conflict of interest to declare. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.foodcont.2017.11.043. References Atmar, R. L., Opekun, A. R., Gilger, M. A., et al. (2014). Determination of the 50% human infectious dose for Norwalk virus. Journal of Infectious Diseases, 209(7), 1016e1022. Bok, K., Abente, E. J., Realpe-Quintero, M., et al. (2009). Evolutionary dynamic of
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GII4 norovirus over a 34-year period. Journal of Virology, 83(22), 11890e11901. Burkhardt, W., 3rd, & Calci, K. R. (2000). Selective accumulation may account for shellfish-associated viral illness. Applied and Environmental Microbiology, 66(4), 1375e1378. Furuya, Y., Katayama, T., Takahashi, T., et al. (2005). Norovirus gastroenteritis in kanagawa prefecture in december 2004. Japanese Journal of Infectious Diseases, 58(6), 391e392. Hall, A. J. (2012). Noroviruses: The perfect human pathogens? Journal of Infectious Diseases, 205(11), 1622e1624. Kageyama, T., Kojima, S., Shinohara, M., et al. (2003). Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J Clin Microbiol, 41(4), 1548e1557. Kroneman, A., Vega, E., Vennema, H., et al. (2013). Proposal for a unified norovirus nomenclature and genotyping. Archives of Virology, 158(10), 2059e2068. Lanata, C. F., Fischer-Walker, C. L., Olascoaga, A. C., et al. (2013). Global causes of diarrheal disease mortality in children <5 years of age: A systematic review. PLoS One, 8(9), e72788. https://doi.org/10.1371/journal.pone.0072788. Le Guyader, F. S., Atmar, R. L., & Le Pendu, J. (2012). Transmission of viruses through shellfish: When specific ligands come into play. Current Opinion in Virology, 2(1), 103e110. Le Guyader, F. S., Loisy, F., Atmar, R. L., et al. (2006). Norwalk virus-specific binding to oyster digestive tissues. Emerging Infectious Diseases, 12(6), 931e936. Le Guyader, F. S., Parnaudeau, S., Schaeffer, J., et al. (2009). Detection and quantification of noroviruses in shellfish. Applied and Environmental Microbiology, 75(3), 618e624. Li, D., Stals, A., Tang, Q. J., et al. (2014). Detection of noroviruses in shellfish and semiprocessed fishery products from a Belgian seafood company. Journal of Food Protection, 77(8), 1342e1347. Loisy, F., Atmar, R. L., Guillon, P., et al. (2005). Real-time RT-PCR for norovirus screening in shellfish. J Virol Methods, 123(1), 1e7. Maalouf, H., Schaeffer, J., Parnaudeau, S., et al. (2011). Strain-dependent norovirus bioaccumulation in oysters. Applied and Environmental Microbiology, 77(10), 3189e3196. Pan, X. (2015). Research progress of norovirus. Journal of Medical Postgraduates, 28(3), 225e228. Patel, M. M., Hall, A. J., Vinje, J., et al. (2009). Noroviruses: A comprehensive review. Journal of Clinical Virology, 44(1), 1e8. Pinto, R. M., Costafreda, M. I., & Bosch, A. (2009). Risk assessment in shellfish-borne outbreaks of hepatitis A. Appl. Environ. Microbiol., 75(23), 7350e7355. Liao, Q. H., Lu, R., Miao, J., et al. (2016). Guidelines on outbreak investigation, prevention and control of norovirus infection (2015). Chinese Preventive Medicine, 50(1), 7e16. da Silva, A. K., Le Saux, J. C., Parnaudeau, S., et al. (2007). Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: Different behaviors of genogroups I and II. Appl Environ Microbiol, 73(24), 7891e7897. Svraka, S., Duizer, E., Vennema, H., et al. (2007). Etiological role of viruses in outbreaks of acute gastroenteritis in The Netherlands from 1994 through 2005. J Clin Microbiol, 45(5), 1389e1394. Teunis, P. F., Moe, C. L., Liu, P., et al. (2008). Norwalk virus: How infectious is it? Journal of Medical Virology, 80(8), 1468e1476. Thornton, A. C., Jennings-Conklin, K. S., & McCormick, M. I. (2004). Noroviruses: Agents in outbreaks of acute gastroenteritis. Disaster Management & Response, 2(1), 4e9. Webby, R. J., Carville, K. S., Kirk, M. D., et al. (2007). Internationally distributed frozen oyster meat causing multiple outbreaks of norovirus infection in Australia. Clinical Infectious Diseases, 44, 1026e1031. Yu, Y., Cai, H., Hu, L., et al. (2015). Molecular epidemiology of oster-related human noroviruses and their global genetic diversity and temporal-geographical distribution from 1983 to 2014. Applied and Environmental Microbiology, 81(21), 7615e7624. Zheng, D. P., Ando, T., Fankhauser, R. L., et al. (2006). Norovirus classification and proposed strain nomenclature. Virology, 346(2), 312e323.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Norovirus contamination in retail oysters from Beijing and Qingdao, China amus Fanning a, b, c, Li Nan a, Wang Jiahui a, Jiang Tao a, Han Chunhui a, Se Zhang Hongyuan a, Zhang Jing a, Li Fengqin a, * a
Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing, China UCD-Centre for Food Safety, School of Public Health, Physiotherapy and Sports Science, University College Dublin, Belfield, Dublin D04 N2E5, Ireland Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, United Kingdom b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 August 2017 Received in revised form 27 November 2017 Accepted 30 November 2017 Available online 5 December 2017
The consumption of raw oyster has been linked to numerous foodborne gastroenteritis outbreaks caused by norovirus (NoV) in different countries of the world, including in China. This study investigated the prevalence of NoV and contamination levels in retail oysters taken from markets in Beijing and Qingdao in China. The oysters were collected monthly from seafood markets between September 2015 and September 2016 (13 months) in both cities. The digestive glands of these oysters were dissected and NoV particles were extracted. Viral RNA was detected using a TaqMan-based real time one-step reverse transcription-PCR (qRT-PCR) protocol. Of total 672 oyster samples assessed, 652 were considered to be valid for inclusion in the study. The prevalence of NoV was 20.71% (135/652), and this included 21.48% (29/135) of the samples that were positive for the GI strain alone, 62.96% (85/135) contained the GII strain alone and 15.56% (21/135) were mixed with GI and GII, respectively. A total of 68 NoV-positive samples were quantified by qRT-PCR and values obtained ranged from 3.55 103 to 1.45 106 genomic copies per g digestive tissue. The NoV contamination in retail oysters fluctuated with the sampling month and peaks of contamination occurred in February (49.12%) and March (55.36%) 2016, respectively. Considering some of the oysters were consumed as raw or half-cooked in China, as a risk reduction measure these oysters should be well heated prior to consumption. Furthermore, successive and region-extended monitoring in retail oysters for NoV as well as risk communication is recommended. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Norovirus oyster contamination realtime reverse transcription-polymerase chain reaction (qRT-PCR)
1. Introduction Norovirus (NoV) is known as the leading cause of human nonbacterial acute gastroenteritis that can infect individuals of all age groups worldwide (Hall, 2012). NoV is a non-enveloped virus with a single-stranded, positive-sense RNA genome. It is belonging to genus Norovirus, family Caliciviridae (Kroneman et al., 2013). NoV genus is currently divided into 6 distinct genogroups (denoted as GI through to GVI), each of which has been subdivided into multiple genotypes (Zheng et al., 2006; Kroneman et al., 2013). GI and GII, can be subdivided into 9 and 22 genotypes respectively,
* Corresponding author. No.7, Panjiayuan Nanli, Chaoyang District, Beijing, 100021, PR China. E-mail address: [email protected] (L. Fengqin). https://doi.org/10.1016/j.foodcont.2017.11.043 0956-7135/© 2017 Elsevier Ltd. All rights reserved.
and both of these constitute the primary strains epidemiologically linked to human cases of acute gastroenteritis (Liao et al., 2016). Norovirus infections constitute a serious disease burden both in developed and developing countries. According to the U.S. Centers for Disease Control and Prevention (US CDC), NoV was responsible for 60% of acute gastroenteritis cases or 21 million cases in the United States each year (Patel et al., 2009). Children, the elderly and immunocompromised persons may experience more severe symptoms and/or extended duration of illness or even chronic diarrhea. There are an estimated 70,000 norovirus-associated deaths recorded among children <5 years annually worldwide (Lanata et al., 2013). In China, reports of acute gastroenteritis cases caused by NoV have been increasing and these now account for 60e96% of nonbacterial infectious diarrhea recorded since 2012 (Liao et al., 2016). Norovirus is mainly transmitted via the fecal-oral route and
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contaminated food is one of the important vehicles for transmission. According to US CDC, of 348 NoV outbreaks reported from January 1996 to November 2000, the sources included food (39%), person-to-person contact (20%) and water (3%) (Thornton, Jennings-Conklin, & McCormick, 2004). Shellfish, especially oysters, are a recognized transmission vector for human NoV in the environment (Yu et al., 2015). These shellfish can filter large volumes of water as part of their filter-feeding activities and consequently accumulate and concentrate the virus in their digestive gland. Furthermore, because oysters are typically grown in coastal water that potentially is contaminated by human waste along with the fact that oysters are often consumed under-cooked or raw, this food matrix presents a high risk for viral infections, frequently being involved in NoV outbreaks (Webby et al., 2007). On the basis of data provided by European Food Alert System for Food and Feed (RASFF), all but 3 of the 36 outbreak notifications involving viruses reported during an 11-year period (2000e2011) were attributed to NoV. Among these, 22 were associated with oysters (Le Guyader, Atmar, & Le Pendu, 2012). In addition, secondary transmission from person-to-person may occur, and outbreaks may give rise to school and workplace closures, as well as the closure of oyster harvesting waters along with recalls. Although the adoption of regulations that specify acceptable levels of bacterial enteric pathogens in shellfish tissues or in shellfish-growing water has significantly decreased the impact of bacteria as causes of shellfishassociated disease outbreaks, these regulations have failed to prevent many outbreaks of viral origin (Le Guyader et al., 2009). China is one of the largest countries in the world for consumption and production of oysters. Little information is available to describe oyster contamination with NoV. To protect consumer health in China, the NoV contamination in retail oysters sourced in Beijing and Qingdao cities were monitored over a 13-month period. Data obtained reported on the quantification of this viral genomic copies in the retail oysters collected. Further the temporal geographical distribution of oyster-related NoV was described in order to extend our knowledge of NoV prevalence in China. 2. Materials and methods 2.1. Oyster sampling A total of 672 fresh oyster samples were randomly collected from one seafood wholesale market in Beijing and in addition six retail markets in Qingdao from September 2015 to September 2016 (covering a period of 13 months). This accounted for approximately 30e40 samples per month. The seafood wholesale market recruited to the study was the biggest establishment in Beijing and oysters from here were placed routinely for sale in many supermarkets or farmer's markets, being dispatched from this location. On each sampling day, no more than 4 retail outlets were selected for study. A maximum of 30 individual oysters were selected randomly for testing from each of these sampling sites. Samples were transported on ice directly to the laboratory and NoV extraction was carried out within 24 h. 2.2. Oyster sample processing Since the weight of the oysters differed depending on the individual oyster or collection season, each sample was adjusted so that it contained 2e5 individual oysters and make sure each sample contains 2 g digestive gland of oyster. The extraction procedure of NoV from digestive gland of oyster was referred to Microbiology of the food chain–Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR (15216-1), but has some changes in number of oysters as one sample. Briefly the shell of
each oyster was carefully washed with tap-water and the digestive glands dissected carefully from other tissues. All digestive glands for these 2e5 oyster samples were pooled and homogenized thoroughly. A portion of 2 g of homogenized digestive tissues was then transferred into a 50-mL centrifuge tube followed by the addition of 10 mL mengovirus VMC0 (CeeramTooLs, France) as the process control and 2 mL proteinase K (Sigma, U.S.A.) solution with a final concentration of 0.1 mg/mL. This mixture was incubated in a shaking incubator at 280 rpm for 60 min at 37 C and then for a further 15 min in a water bath at 60 C. After centrifugation at 3000 rpm for 5 min at room temperature, the supernatant (approximately 3.0 mL) was collected into a sterile Eppendorf tube and stored at 80 C until required. 2.3. NoV nucleic acid purification Viral RNA was extracted and purified using a QIAamp Viral RNA Mini kit (QIAgen, Hilden, Germany) in a final volume of 50 mL, in accordance with the manufacturer's instructions. The extracted RNA were then used as the template for real time reverse transcription polymerase chain reaction (qRT-PCR) and maintained at 80 C prior to analysis for not longer than one week. 2.4. Real-time RT-PCR amplification Oligonucleotide primers and detection probes employed in this study to identify GI, GII and along with the mengovirus were shown in Table 1. The real-time RT-PCR was carried out using RNA Ultrasense One-step Quantitative RT-PCR System (Invitrogen, U.S.A.). All samples extracted were screened for NoV GI, GII and mengovirus by triplex qRT-PCR, performed in a CFX96 (BIO-RAD,U.S.A.) real-time PCR platform. The GI probe was labelled with 5-hexachloro-fluorescein (HEX) at 50 -end and black hole quenching1(BHQ1) at 30 -end and be detected at 556 nm fluorescence channel. The GII probe was labelled with 6-carboxyfluorescein (FAM) at 50 -end and black hole quenching1(BHQ1) at 30 -end and be detected at 515 nm fluorescence channel. The mengovirus probe was labelled with 5-Nhydroxysuccinimide ester (CY5) at 50 -end and black hole quenching2(BHQ2) at 30 -end and be detected at 667 nm fluorescence channel. The three different gene fragments can be detected simultaneously through different fluorescence channel in each well. The final reaction volume of 20 mL (without nucleic acid template) consisted of 500 nM of each upstream primer; 900 nM of each downstream primer; 250 nM of each detection probe, along with buffer and enzymes at concentrations recommended by the manufacturer. 5 mL of purified nucleic acid or control was added per well, and the final volume in each well was 25 mL. The thermal profile comprised 55 C for 30 min and 95 Cfor 5 min, followed by 45 cycles of 95 C for 15s, 60 C for 1min and 65 C for 1 min. Additionally, highly purified water was used as the blank qRT-PCR control. Phosphate-buffered saline (0.01 mol/L, pH7.2) was used as the negative process control and was run through all stages of the analytical process. The cycle threshold (Ct) was set manually at 0.1, and it was always on the logarithmic portion of the amplification curve thereby being distinguishable from the background fluorescence. Those samples for which reactions yielded a Ct value below or equal to 35 for NoV GI or GII were considered as positive, and the Ct value above or equal to 38 for NoV GI or GII were considered as negative, and the Ct value between 35 and 38, qRT-PCR detection should be repeated. 2.5. Extraction efficiency To control for false-negative signals due to extraction failure, a
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Table 1 Primers and Probes for NoV detection used in the present study. Name GI
GII
Mengo virus
QNIF4(FW) NV1LCR (REV) NVGG1P(PROBE) QNIF(FW) COG2R (REV) QNIFs(PROBE) Mengo110(FW) Mengo209 (REV) Mengo147(PROBE)
Primers and Probes
reference
50 -CGC TGG ATG CGN TTC CAT-30 50 -CCT TAG ACG CCA TCA TCA TTT AC-30 50 -HEX-TGG ACA GGA GAY CGC RAT CT-BQ1-30 50 -ATG TTC AGR TGG ATG AGR TTC TCW GA-30 50 -TCG ACG CCA TCT TCA TTC ACA-30 50 -FAM-AGC ACG TGG GAG GGC GAT CG-BQ1-30 50 -GCG GGT CCT GCC GAA AGT-30 50 -GAA GTA ACA TAT AGA CAG ACG CAC AC-30 50 -CY5-ATCACA TTA CTG GCC GAA GC-BQ2-30
da Silva et al., 2007 Svraka et al., 2007 Svraka et al., 2007 Loisy et al., 2005 Kageyama et al., 2003 Loisy et al., 2005 Pinto, Costafreda, & Bosch, 2009 Pinto et al., 2009 Pinto et al., 2009
process control (mengovirus) was included in each sample. The extraction efficiency of the spiked mengovirus from an individual sample was calculated using the following equation: extraction efficiency (%)¼(the number of recovered mengovirus genomic copies/the number of seeded mengovirus genomic copies) 100%. Samples with the Ct value of GI or GII NoV above or equal to 38 and the extraction efficiency below 1% should be discarded.
2.6. Sensitivity and standard curves of GI and GII NoV The sensitivity and standard curves of GI and GII NoV were determined using a Norovirus GI Q and Norovirus GII Q Standard kit (CeeramTooLs, France) with real-time RT-PCR. A standard curve of GI or GII was generated using a 10-fold series of dilutions ranging from 104 to 101 viral genomic copies per microliter and a 2-fold series of dilutions ranging from 10 to 1.25 viral genomic copies per microliter. Viral genomic copy numbers contained in each of the analyzed samples were determined by comparison with the standard curve. The Norovirus GI Q Standard and Norovirus GII Q Standard were also used as positive control.
3. Results 3.1. Sensitivity and standard curves of GI and GII NoV The sensitivity of the method is 2.5 viral genomic copies per microliter (or 12.5 viral genomic copies per reaction) for GI and GII, and both of them corresponding Ct values were below 38. The limit of quantification was 10 genomic copies per microliter (or 50 viral genomic copies per reaction) for GI and GII. The standard curves of GI and GII were shown in Fig. 1.
3.2. NoV prevalence in retail oysters Among 672 oyster samples analyzed, 20 were determined to have a Ct value above 38 for both GI and GII NoV and thus were excluded as their extraction efficiency was unacceptable (less than 1%). Data obtained from 652 samples were considered to be valid. A total of 135 (20.70%, 135/652) samples were contaminated with NoV, 55 positives were recorded from Beijing and 80 from Qingdao, respectively. Genogrouping data for these 135 NoV-positive samples revealed that 29 (21.48%, 29/135) samples were identified as being contaminated with GI NoV alone, 85 (62.96%, 85/135) by GII NoV alone and 21 (15.56%, 21/135) were mixtures of GI and GII NoV. The prevalence of NoV in retail oysters from different regions was shown in Table 2. In terms of NoV contamination in oysters from Beijing, 55 (16.03%, 55/343) samples were positive for NoV, comprising 12 (21.82%, 12/55) GI strains alone, 37 (67.27%, 37/55) GII strains alone and 6 (10.91%, 6/55) co-contaminated with both GI and GII strains. Comparatively, samples from Qingdao, a coastal city, were more heavily contaminated with norovirus (80/309, 25.89%) than those from Beijing (p ¼ 0.002). The prevalence of NoV genogroups identified in samples taken from Qingdao was 21.25% (17/80) for GI alone, 60.00% (48/80) for GII alone, and 18.75% (15/ 80) for both, respectively. No significant difference in prevalence of any genogroups of NoV detected from oysters between the two cities was observed. 3.3. NoV seasonality The prevalence of NoV in retail oysters varied with the sampling seasons. Contamination appeared to peak in February (49.12%) and March (55.36%); in 2016. A peak of oyster contamination by NoV in the cool and dry months of the year was observed in Beijing (February, 42.22%) and Qingdao (March, 78.12%), respectively. As Qingdao is a coastal city with a large oyster production industry together with high levels of consumption, NoV contamination were also frequently detected in the late autumn with 35.29% of recorded positives occurring in October and 46.88% for November, respectively. No NoV was detected in oysters collected in August 2016 in both cities. Table 3 showed the oyster contamination with NoV as it varied with sampling time. 3.4. Quantification of NoV in naturally contaminated oysters
Fig. 1. Standard curves for real-time RT-PCR detection of GI and GII, respectively.
The number of viral genomic copies in oysters was calculated by multiplying the value generated from the real-time RT-PCR by the dilution factors from the viral and RNA extraction. Accordingly, the limit of quantification (LOQ) was 3.33 103 genomic copies per gram of digestive tissues (DT) for NoV GI and GII. Only those samples wherein the extraction efficiency was equal to or higher than 10% were assigned to perform a quantitative analysis for NoV. A total of 68 samples were therefore available for NoV
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Table 2 Norovirus prevalence in retail oysters from Beijing and Qingdao. Region
Beijing Qingdao Total
No. of samples
343 309 652
No. of positive samples (%)
55 (16.03%) 80 (25.89%) 135 (20.71%)
NoV genogroup No. of GI positive samples (%)
No. of GII positive samples (%)
No. of GI & GII positive samples (%)
12 (21.82%) 17 (21.25%) 29 (21.48%)
37 (67.27%) 48 (60.00%) 85 (62.96%)
6 (10.91%) 15 (18.75%) 21 (15.56%)
Table 3 Comparison of NoV contamination in retail oysters in different months. Month
Sep 2015 Oct 2015 Nov 2015 Dec 2015 Jan 2016 Feb 2016 Mar 2016 Apr 2016 May 2016 Jun 2016 Jul 2016 Aug 2016 Sep 2016
Total Samples
43 59 54 47 34 57 56 77 49 42 42 43 49
Positive Samples (Rate)
4 (9.30%) 12 (20.34%) 18 (33.33%) 2 (4.26%) 4 (11.76%) 28 (49.12%) 31 (55.36%) 6 (7.79%) 9 (18.37%) 8 (19.05%) 2 (4.76%) 0 (0.00%) 11 (22.45%)
Beijing
Qingdao
Total Samples
Positive Samples (Rate)
Total Samples
Positive Samples (Rate)
19 25 22 16 23 45 24 52 24 18 24 27 24
4 (21.05%) 0 (0.00%) 3 (13.64%) 2 (12.50%) 2 (8.70%) 19 (42.22%) 6 (25.00%) 6 (11.54%) 0 (0.00%) 6 (33.33%) 0 (0.00%) 0 (0.00%) 7 (29.17%)
24 34 32 31 11 12 32 25 25 24 18 16 25
0 (0.00%) 12 (35.29%) 15 (46.88%) 0 (0.00%) 2 (18.18%) 9 (75.00%) 25 (78.12%) 0 (0.00%) 9 (36.00%) 2 (8.33%) 2 (11.11%) 0 (0.00%) 4 (16.00%)
quantification. The numbers of GI NoV in these oysters ranged from 3.55 103 to 1.70 104copies/g DT (geometric mean: 4.98 103 copies/g DT), and GII NoV between 5.04 103 and 1.45 106copies/ g DT (geometric mean: 7.04 104 copies/g DT) (Table 4). The highest quantities of NoV copies for GI and GII in oysters were found in April 2016 and October 2015, respectively.
4. Discussion Shellfish production is one of the most important economic activities in coastal areas of China. Most oysters produced are consumed as raw or half-cooked and this feature of consumption presents a high risk for viral infections, in humans, often leading to NoV outbreaks (Yu et al., 2015). This study was focused on the prevalence and contamination levels of detectable NoV in oysters, available for purchase in major population centers in China. For NoV detection, a sensitive and reproducible method was required. At present, the methods for NoV detection include electron microscopy (or immune electron microscopy), enzyme immune assay and molecular methods. qRT-PCR with high sensitivity and specificity was considered to be a useful method for the detection of NoV in foods (Pan, 2015). In this study a tailored qRTPCR method for NoV detection was developed and used in the survey of NoV in oysters. As NoV was difficult to culture in vitro and we cannot get enough NoV particles with definite number to spike sample, so the limit of detection (LOD) and the limit of
quantification (LOQ) were more theoretical LOD and LOQ. The result maybe overestimate the sensitivity of the method and underestimated the content of NoV genomic copies in samples. Beijing is an international metropolis. Foods produced all over the country and many part of the world can be consumed in Beijing. In contrast, Qingdao is a coastal city in northern China and oyster farming is one of the important economic activities. Both of these cities were considered as useful sampling pools for oysters potentially contaminated with NoV. The results of this study showed that the average prevalence of NoV in retail oysters was 20.71% a figure that fluctuates with the sampling season. The contamination rate in winter was higher than that in summer. It was reported previously that oyster glycogen played an important role in NoV binding and it is present at high levels in the flesh of shellfish from November through March (Furuya et al., 2005; Le Guyader et al., 2006; Burkhardt and Calci, 2000), a period coinciding with seasonal outbreaks of NoV. The prevalence of NoV GII(62.96%) was higher than that of GI(21.48%) in the retail oysters and only 21 samples (15.56%) were co-infected by GI and GII. This result differed from observations published earlier wherein it was reported that GI NoV was detected more often than GII in shellfish. (Maalouf et al., 2011; Li et al., 2014). Previously it was suggested that the probability of NoV infection was dose dependent (Bok et al., 2009) and the infectious dose was suggested to be about 18-2800 infectious virus particles (Teunis et al., 2008; Atmar et al., 2014). In this study, the number of NoV
Table 4 Quantification of NoV contamination in oysters. NoV genotype
No. of samples
Geometric concentration of GI NoV (copies/g DT) maximum
GI GII GIþGII
15 51 2
4
1.70 10 e 6.80 103
minimum 3
3.55 10 e 3.79 103
Geometric concentration of GII NoV (copies/g DT) geometric mean 3
4.98 10 e 5.08 103
maximum
minimum
geometric mean
e 1.45 106 1.28 104
e 5.04 103 8.83 103
e 7.04 104 1.06 104
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was determined by genomic copies. The data in this study may overestimate the risk of oyster contamination and human infections, as the latter strategy does not relate to the infectious dose. Further, quantification of NoV is regarded as challenging as it may produce considerable variation in results generated by different researchers because of different PCR parameters, reagents, NoV standards (e.g., double-stranded DNA plasmids or synthesized RNA) and even different machines. Therefore, NoV prevalence data and the detection methods used in the specific study must be considered when evaluating results. In summary, a high prevalence of NoV contamination was observed in oysters placed on the retail market for sale and the contamination levels of NoV may result in gastroenteritis symptoms in consumers. Considering some oysters were consumed as raw or lightly-cooked, from a public health view point, it suggests that as a risk reduction measure, consumers should be encouraged to cook oysters. Results of this study will provide the basic information required for risk assessment on dietary exposure to NoV via oysters, and these data will assist policy-makers to develop strategies to limit NoV contamination of oysters and its transmission to humans. Surveillance of NoV contamination in marketed oysters is highly recommended, as a means of extending the epidemiological data available to describe this risk to human health. Additionally, public education and risk communication is the key to prevent NoV gastroenteritis, in the future. Funding This work was supported by the Beijing Natural Science Foundation (grant number: 5141002). Conflicts of interest All authors listed in the manuscript contributed to conception, acquisition, analysis and interpretation of data, design of the manuscript, critically revised the manuscript and approved the final submitted version. The authors have no conflict of interest to declare. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.foodcont.2017.11.043. References Atmar, R. L., Opekun, A. R., Gilger, M. A., et al. (2014). Determination of the 50% human infectious dose for Norwalk virus. Journal of Infectious Diseases, 209(7), 1016e1022. Bok, K., Abente, E. J., Realpe-Quintero, M., et al. (2009). Evolutionary dynamic of
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