Risk of Norovirus Transmission Linked to the Consumption of Raw Vegetables
ILSI SEA Region 6th Asian Conference on Food and Nutrition Safety (Nov 2012) http://www.ilsi.org/SEA_Region/Pages/ViewEventDetails.aspx?WebId=4D540914-EEB6-40E4-89EB-0B73BA3D76C1&ListId=478BE3CB-581B-4BA2-A280-8E00CCB26F9C&ItemID=66
Tuan Zainazor Tuan Chilek, Cheah Yoke Kqueen and Son Radu
University Putra MalaysiaEmail: [email protected]
� Noroviruses (Genus Norovirus, Family Caliciviridae) are a genetically diverse group of single-stranded RNA.
� Small round structured Non-enveloped viruses that cause acute gastroenteritis (an inflammation
NOROVIRUS
that cause acute gastroenteritis (an inflammation of the stomach and intestines) in humans.
� Noroviruses are named after the original strain “Norwalk virus,” which caused an outbreak of gastroenteritis in a school in Norwalk, Ohio, in 1968.
Noroviruses can be classified into five major groups:�Prototype Norwalk virus (GI) - human�Prototype Snow Mountain Agent (GII)
Classification of Norovirus
– human and swine�Prototype Bovine Enteric Calicivirus (GIII)
- bovine�Prototype Alphatron and Fort Lauderdale
virus (GIV) – human �Prototype Murine Norovirus (GV) - murine
(D’Souza and Jaykus, 2006)
� These viruses are transmitted mainly via fecal–oral route through person-to-person contact or consumption of contaminated food, hospital, cruise ship, prison, nursing home, age-care homes, holiday camp, caterers (Japan reported 19% identified to caterer as asymptomatic carriers)
� Fruits and vegetables contamination with enteric virus can occur before the product reaches food service establishment
Transmission of enteric (Norovirus) virus
Transmission of enteric viruses;Koopmans M, Duizer E (2004), Int J Food Microbiol 90:23-41.
Detection of Norovirus
� Today, Noroviruses are recognized as one of the most common cause of infectious gastroenteritis among persons of all ages. For example, they are responsible for <50% of all foodborne gastroenteritis outbreaks in the United States.
� Characterization and classification of Norovirus based on reverse transcription-polymerase chain reaction (RT-PCR), sequencing and phylogenetic analysis can be carried out in standard laboratory.phylogenetic analysis can be carried out in standard laboratory.
� For example, in stool specimens, RT-PCR had the ability to detect 102-104 viral particles/ ml (CDC, 2001).
WHY RAW VEGETABLES?
� Salad vegetables are one of the popular dishes among Malaysian population, usually eaten raw in their meals
� Food samples examined for food poisoning � Food samples examined for food poisoning and outbreak investigation are normally free from pathogenic organism (bacteria) even though the consumers have acute gastroenteritis.
� Therefore, virus could be a suspected agent, especially Norovirus which is commonly associated with acute gastroenteritis.
Bacteria
30%
Percentage of estimated foodborne illness attributable by agent
Protozoan 3%
Viral
67%
*Mead et al., 1999
Hazard Est. Cases DeathsNorwalk virus 23,000,000 naCampylobacter 2,453,926 0.1%Salmonella 1,412,498 0.8%
Foodborne Hazards
Salmonella 1,412,498 0.8%C. perfringens 248,520 0.05%S. aureus 185,060 0.02%E. coli O157:H7 73,480 0.83%L. monocytogenes 2,518 20%C. botulinum 58 8.6%
(Centers for Disease Control and Prevention, 2001)
Route Cases Reference
Food 6,900,000 Mead et al., 1999
Recreational 6,900,000 U.S. Census, 2000
Estimated number of cases of Norovirus per year in United States
Recreational Water
6,900,000 U.S. Census, 2000
Drinking Water
3,584,000 Haas et al., 1999
Others 5,616,000 Mead et al., 1999
Total 23,000,000
SCOPE
� To establish standard methodology in detecting of Norovirus in raw vegetables using RT-PCR Technique
� To provide the baseline data for Norovirus contamination in raw vegetables at retail level
To characterize the Genogroup of Norovirus using RT-� To characterize the Genogroup of Norovirus using RT-PCR and sequencing
Method optimization to obtain positive control from clinical specimens
- Stools- Vomitus- Throat swab
-Viral RNA extracted usingQIAamp ViralRNA Kit (QIAGEN)
-Reverse Transcription (50oC, 30 min)-Initial PCR (95oC,15 min)-Denaturation (94oC,45 sec)-Annealing (52oC,30 sec)-Extending (72oC,45 sec)-40 cycles
SAMPLES RNA EXTRACTIONNOROVIRUS DETECTION
Norovirus genogroup
Primer Sequence Amplicon size
I MON 432 5’ TGG ACI CGY GGI CCY AAY CA 3’ 213 bp
MON 434 5’ GAA SCG CAT CCA RCG GAA CAT 3’
II MON 431 5’ TGG ACI AGR GGI CCY AAY CA 3’ 213 bp
MON 433 5’ GAA YCT CAT CCA YCT GAA CAT 3’
DEVELOPMENT OF NOROVIRUS DETECTION METHOD IN RAW VEGETABLES
Sample collection and preparation
Detachment of viruses from food surfaceDetachment of viruses from food surface
Enhance Concentration of RNAFIVE MAIN STEPS
Viral Extraction
Detection using one-step RT-PCR
English Name Local Name Scientific Name
Indian pennywort Pegaga Centella asiatica
Water spinash Kangkung Ipomoea aquatica
Mung bean sprout Tauge Vigna radiata
Vietnamese Kesum Poligonum minus
Sample collection and preparation
Vietnamese coriander
Kesum Poligonum minus
Japanese parsley Selom Oenanthe stolonifera
Wild cosmos Ulam raja Cosmos caudatus
Celery Daun sup/daun saderi Apium graveolens
Green onion Daun bawang Allium cepa
Indian pennywort
Celery
Green onion
Mung bean sprout
Water spinashVietnamese coriander
Japanese parsleyWild cosmos
- using Tryptose Phosphate Broth Glycine buffer pH 9.0 (TPBG)
25 g sample (about 2-3 cm)
TPBG (50 mL)
25 mM MgCl2 (500 µL)
Detachment of viruses from food surface
25 mM MgCl2 (500 µL)
Shake for 15 mins
Transfer into 50 mL sterile tube
Centrifuge at 4000 rpm for 10 mins
- using negative charge membrane filter
Filter (negative charge membrane filter)
Rinse with H SO (8 mL)
Transfer liquid into 12 mL test tube
Enhance Concentration of RNA
Rinse with H2SO4 (8 mL)
Transfer filter in sterile petri dish
Put TPBG (6 mL)
Agitate at 60 rpm, 15 mins
Adjust pH with 1N HCl (pH7)
Centrifuge at 4000 rpm for 1 hr
- using Qiagen RNeasy mini kit
Transfer 150 µL into 2 mL microtube
Add 15 µL of 10% SDS
Add 450 µL of RLT-ß-mercaptoethanol solution
Vortex for 15 sec.
Viral Extraction
Put protenase K (0.75 µL)
Incubate for 2 mins (RT)
Incubate for 1 hr at 37oC
Heat for 2 mins (56oC)
Incubate at RT for 5 mins
Extract using Qiagen RNeasy mini kit
RNA solution
Reverse transcriptive condition
30 mins 50oC
Initial PCR inactivation 15 mins 95oC
Amplification conditionsMaster Mixture preparation
PCR Conditions
- using One-step RT-PCR
Detection using one-step RT-PCR
Components Volume per reaction
RNase free water 10.0 µL
5x Qiagen One-step RT-PCR 5.0 µL Initial PCR inactivation step
15 mins 95oC
3-step cycling :DenaturationAnnealingExtensionFinal extension
45 sec30 sec45 sec10 mins
94oC52oC72oC72oC
Number of cycles : 40
5x Qiagen One-step RT-PCRbuffer
5.0 µL
dNTP mix (containing 10 mM of each dNTP)
1.0 µL
Forward primer (MON 431 or MON432)
1.5 µL
Reverse primer (MON 433 or MON434)
1.5 µL
Qiagen one-step RT-PCR enzymemix
1.0 µL
Template RNA 5.0 µL
Final volume 25.0 µµµµL
NOROVIRUS CONTAMINATION IN RAW VEGETABLES AT RETAIL LEVEL
Local Name No of Samples Total
Wet market
Night market Grocery store
Supermarket
Sampling of raw vegetables from selected selling point
Pegaga 16 16 16 16 64
Kangkung 16 16 16 16 64
Tauge 16 16 16 16 64
Kesum 16 16 16 16 64
Selom 16 16 16 16 64
Ulam raja 16 16 16 16 64
Daun sup 16 16 16 16 64
Daun bawang 16 16 16 16 64
Total 512
RELATEDNESS OF DIFFERENT NOROVIRUS STRAINS USING GENOMIC SEQUENCING
� Gel Extraction� Cloning – QIAGEN PCR Cloning Kit� Plasmid purification – QIAprep � Plasmid purification – QIAprep
Miniprep Kit (QIAGEN)� Sequence comparison – BLAST� Sequence alignment� Phylogenetic tree
RESULTS
Type of sample ID No. Results
GI GII
Stool S1 – S10 Not Detected Not Detected
Vomitus V1 – V5 Not Detected Not Detected
Results of Norovirus detection in clinical specimens
Vomitus V1 – V5 Not Detected Not Detected
Throat Swab
T1 – T2 Not Detected Not Detected
T3 Detected Not Detected
T4 – T9 Not Detected Not Detected
T10 Detected Not Detected
Representative of agarose gel of Norovirus genogroup I detection in Throat
swab sample (T1-T10). NC, negative control; M, 100bp marker
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 M NC
200bp
300bp
100bp
900bp
213bp
1000bp
Representative agarose gel for Norovirus genogroup I detection in Vietnamese Coriander (1-8). PC (positive control); NC, negative control; M, 100bp marker
Results of method development using positive and negative control
Sample name No. of sample
No. of positive sample Positive control(+)
Negative control(-)
Genogroup I
Genogroup II
Mung bean sprout(Tauge)
10 1 0 + -
Water spinach(Kangkung)
10 0 0 + -
Vietnamese coriander (Kesum)
10 1 0 + -
Japanese parsley(Selom)
10 0 0 + -
Wild cosmos(Ulam raja)
10 0 0 + -
Indian pennywort(Pegaga)
10 1 0 + -
Celery(Daun Sup)
10 1 0 + -
Green onion(Green onion)
10 1 0 + -
Type of samples No of samples Positive(Genogroup I)
Prevalence (%)
Pegaga 64 6 9.37
9.38Kangkung 64 4
Tauge 64 10 15.62
Prevalence data of Norovirus Genogroup I in raw vegetables
Tauge 64 10 15.62
12.50Kesum 64 8
Selom 64 6 9.37
0Ulam raja 64 0
Daun sup 64 8 12.50
12.50Daun bawang 64 8
9.37 9.38
15.62
12.5
9.37
12.5 12.5
6
8
10
12
14
16
Pre
vale
nce
(%
)
Incidence of Norovirus GI in Salad Vegetables
Pegaga
Kangkung
Tauge
Kesom
Selom
0
0
2
4Pre
vale
nce
(%
)
Pegag
aKan
gkun
gTau
geKes
omSelo
mUlam R
ajaDaun
Sup
Daun B
awang
Types of sample
Ulam Raja
Daun Sup
Daun Bawang
* Norovirus Genogroup II was not detected from all samples
10
15
20
25
To
tal P
reva
len
ce (
%)
Norovirus genogroup I detection in raw vegetables from selected sampling location
Grocery Stores
Prevalence data of Norovirus Genogroup I detection in raw vegetables from selected sampling location.
0
5To
tal P
reva
len
ce (
%)
Mung bean sprout
Water spinash
Vietnamese coriander
Japanese parsley
Wild cosmos
Indian pennywortCelery
Green onion
Types of Raw Vegetables
Wet Market
Night Market
Supermarket
Observation during sampling
The condition of raw vegetables display at the selected grocerystores during sampling.stores during sampling.
The condition of raw vegetables display at the selected supermarketduring sampling.
Norovirus Pegaga TGGACGCGCGGGCCTAATCATTCAGATCCATCAGAGACTCTAGTGCCACA 50Norovirus Kangkung TGGACGCGTGGGCCTAATCATTCAGATCCATCAGAGACTCTAGTGCCACA 50Norovirus Tauge TGGACGCGTGGGCCTAACCATTCAGATCCATCAGAGACTCTAGTGCCACA 50Norovirus Kesum TGGACGCGTGGGCCCAATCATTCAGATCCATCAGAGACTCTAGTGCCACA 50
**************************************************
Norovirus Pegaga CACTCAAAGAAAA-TACAGTTGATTTCACTTCTAGGGGAAGCTTCACTCC 99Norovirus Kangkung CACTCAAAGAAAAATACAGTTGATTTCACTTCTAGGGGAAGCTTCACTCC 100Norovirus Tauge CACTCAAAGAAAAATACAGTTGATTTCACTTCTAGGGGAAGCTTCACTCC 100Norovirus Kesum CACTCAAAGAAAAATACAGTTGATTTCACTTCTAGGGGAAGCTTCACTCC 100
*************************************************
Relatedness of different Norovirus strains using genomic sequencing�Sequence Alignment
Norovirus Pegaga ATGGTGAGAAATTTTACAGAAAGATTTCCAGCAAGGTCATACATGAAATC 149Norovirus Kangkung ATGGTGAGAAATTTTACAGAAAGATTTCCAGCAAGGTCATACATGAAATC 150Norovirus Tauge ATGGTGAGAAATTTTACAGAAAGATTTCCAGCAAGGTCATACATGAAATC 150Norovirus Kesum ATGGTGAGAAATTTTACAGAAAGATTTCCAGCAAGGTCATACATGAAATC 150
**************************************************
Norovirus Pegaga AAGACTGGTGGATTGGAAATGTATGTCCCAGGATGGCAGGCCATGTTCCG 199Norovirus Kangkung AAGACTGGTGGATTGGAAATGTATGTCCCAGGATGGCAGGCCATGTTCCG 200Norovirus Tauge AAGACTGGTGGATTGGAAATGTATGTCCCAGGATGGCAGGCCATGTTCCG 200Norovirus Kesum AAGACTGGTGGATTGGAAATGTATGTCCCAGGATGGCAGGCCATGTTCCG 200
**************************************************
Norovirus Pegaga TTGG-ATGCGGTTAT----- 213Norovirus Kangkung TTGG-ATGCGGTTATCTT-- 217Norovirus Tauge TTGG-ATGCGGTTC------ 213Norovirus Kesum TTGG-ATGCGATCT------ 213
**************************
GI
Phylogenetic analysis of Norovirus strains based on RNA-dependent RNA-polymeraseregion (RdRp); Tree-Neighbour joining using BLOSUM62. The phylogenetic tree is
based on sequences from different types of raw vegetables collected in this study.
Conclusion
� The first kind of study to detect the present of Norovirus in raw vegetables in Malaysia
� RT-PCR method is an excellent choice for the detection of Norovirus
� It is evident that the NorovirusGenogroup 1 contributes to the acute gastroenteritis diseases in Malaysia (2 of the 10 throat swabs samples have been detected positive for the presence swabs samples have been detected positive for the presence of Norovirus)
� Norovirus Genogroup 1 was detected from salad vegetables which is common in human infection
� This result highlights the risk associated with the consumption of raw vegetables contaminated with Norovirus and that raw vegetables could be a medium or transmission vehicle of Norovirus
� Avoid contamination of Norovirus – eliminate/reduce through proper washing and handling to detach viral particle from salad vegetables surfaces
Output from this study
� Norovirus detection method was established
� Baseline data on Norovirus made available
� Proposed this methodology to Ministry of Health � Proposed this methodology to Ministry of Health Malaysia
� Training for MOH food laboratory staff
� National monitoring program (year 2012)
� Proposed guidelines on Norovirus contamination
Training
� NPHL, Sungai Buloh - Lab staff(7 March 2012).
� Starcruise, Penang – Cabin crew (29 June 2011).
� Unisel, Shah Alam – Workshop participant (17 Jan 2011 & 24 Jan 2011).
� Food Quality and Safety Division – Financial Support� National Public Health Laboratory, Sungai Buloh� University Putra Malaysia
ACKNOWLEDGEMENTS