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: son@putra.upm.edu.my

� 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