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 Risk of Norovirus Transmission Linked to the Consumption of Raw Vegetables Tuan Zainazor Tuan Chilek, Cheah Yoke Kqueen and Son Radu University Putra Malaysia Email: son@putra.upm.edu.my
NOROVIRUS 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 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.
Classification of Norovirus Noroviruses can be classified into five major groups: Prototype Norwalk virus (GI) - human Prototype Snow Mountain Agent (GII) 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)
Transmission of enteric (Norovirus) virus 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 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. For example, in stool specimens, RT-PCR had the ability to detect 10 2-10 4 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 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.
Percentage of estimated foodborne illness attributable by agent Bacteria 30% Viral 67% Protozoan 3% *Mead et al., 1999
Foodborne Hazards Hazard Est. Cases Deaths Norwalk virus 23,000,000 na Campylobacter 2,453,926 0.1% 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)
Estimated number of cases of Norovirus per year in United States Route Cases Reference Food 6,900,000 Mead et al., 1999 Recreational 6,900,000 U.S. Census, 2000 Water 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- PCR and sequencing
Method optimization to obtain positive control from clinical specimens - Stools - Vomitus - Throat swab -Viral RNA extracted using QIAamp Viral RNA Kit (QIAGEN) -Reverse Transcription (50 o C, 30 min) -Initial PCR (95 o C,15 min) -Denaturation (94 o C,45 sec) -Annealing (52 o C,30 sec) -Extending (72 o C,45 sec) -40 cycles SAMPLES RNA EXTRACTION NOROVIRUS 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 surface FIVE MAIN STEPS Enhance Concentration of RNA Viral Extraction Detection using one-step RT-PCR
Sample collection and preparation English Name Local Name Scientific Name Indian pennywort Pegaga Centella asiatica Water spinash Kangkung Ipomoea aquatica Mung bean sprout Tauge Vigna radiata 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
Celery Mung bean sprout Indian pennywort Green onion Vietnamese coriander Water spinash Wild cosmos Japanese parsley
Detachment of viruses from food surface - using Tryptose Phosphate Broth Glycine buffer ph 9.0 (TPBG) 25 g sample (about 2-3 cm) TPBG (50 ml) 25 mm MgCl 2 (500 µl) Shake for 15 mins Transfer into 50 ml sterile tube Centrifuge at 4000 rpm for 10 mins
Enhance Concentration of RNA - using negative charge membrane filter Filter (negative charge membrane filter) Rinse with H 2 SO 4 (8 ml) Transfer filter in sterile petri dish Put TPBG (6 ml) Transfer liquid into 12 ml test tube Adjust ph with 1N HCl (ph7) Centrifuge at 4000 rpm for 1 hr Agitate at 60 rpm, 15 mins
Viral Extraction - using Qiagen RNeasy mini kit Transfer 150 µl into 2 ml microtube Add 450 µl of RLT-ßmercaptoethanol solution Add 15 µl of 10% SDS Vortex for 15 sec. Put protenase K (0.75 µl) Heat for 2 mins (56 o C) Incubate for 2 mins (RT) Incubate at RT for 5 mins Incubate for 1 hr at 37 o C Extract using Qiagen RNeasy mini kit RNA solution
PCR Conditions Detection using one-step RT-PCR - using One-step RT-PCR Master Mixture preparation Components Volume per reaction RNase free water 10.0 µl Amplification conditions Reverse transcriptive condition 30 mins 50 o C 5x Qiagen One-step RT-PCR 5.0 µl buffer dntp mix (containing 10 mm of each dntp) Forward primer (MON 431 or MON 432) Reverse primer (MON 433 or MON 434) Qiagen one-step RT-PCR enzyme mix 1.0 µl 1.5 µl 1.5 µl 1.0 µl Template RNA 5.0 µl Final volume 25.0 µl Initial PCR inactivation step 3-step cycling : Denaturation Annealing Extension Final extension Number of cycles : 40 15 mins 95 o C 45 sec 94 o C 30 sec 52 o C 45 sec 72 o C 10 mins 72 o C
NOROVIRUS CONTAMINATION IN RAW VEGETABLES AT RETAIL LEVEL Sampling of raw vegetables from selected selling point Local Name No of Samples Total Wet market Night market Grocery store Supermarket 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 Miniprep Kit (QIAGEN) Sequence comparison BLAST Sequence alignment Phylogenetic tree
RESULTS Results of Norovirus detection in clinical specimens Type of sample ID No. Results GI GII Stool S1 S10 Not Detected Not Detected Vomitus V1 V5 Not Detected Not Detected T1 T2 Not Detected Not Detected Throat Swab 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 1000bp 900bp 300bp 213bp 200bp 100bp
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 Genogroup I Genogroup II Positive control (+) Negative control (-) Mung bean sprout (Tauge) Water spinach (Kangkung) Vietnamese coriander (Kesum) Japanese parsley (Selom) Wild cosmos (Ulam raja) Indian pennywort (Pegaga) Celery (Daun Sup) Green onion (Green onion) 10 1 0 + - 10 0 0 + - 10 1 0 + - 10 0 0 + - 10 0 0 + - 10 1 0 + - 10 1 0 + - 10 1 0 + -
Prevalence data of Norovirus Genogroup I in raw vegetables Type of samples No of samples Positive (Genogroup I) Prevalence (%) Pegaga 64 6 9.37 Kangkung 64 4 9.38 Tauge 64 10 15.62 Kesum 64 8 12.50 Selom 64 6 9.37 Ulam raja 64 0 0 Daun sup 64 8 12.50 Daun bawang 64 8 12.50
Incidence of Norovirus GI in Salad Vegetables 15.62 12.5 9.37 9.38 9.37 0 12.5 12.5 16 14 12 10 8 6 4 2 0 Pegaga Kangk ung Tauge Kesom Selom Ulam Raja Daun Sup Daun Bawang Prevalence (%) Pegaga Kangkung Tauge Kesom Selom Ulam Raja Daun Sup Daun Bawang Types of sample * Norovirus Genogroup II was not detected from all samples
Norovirus genogroup I detection in raw vegetables from selected sampling location 25 Tota al P revalence (% ) 20 15 10 5 0 Mung bean sprout Water spinash Vietnamese coriander Japanese parsley Wild cosmos Indian pennywort Celery Types of Raw Vegetables Green onion Grocery Stores Wet Market Night Market Supermarket Prevalence data of Norovirus Genogroup I detection in raw vegetables from selected sampling location.
Observation during sampling The condition of raw vegetables display at the selected grocery stores during sampling. The condition of raw vegetables display at the selected supermarket during sampling.
Relatedness of different Norovirus strains using genomic sequencing Sequence Alignment Norovirus Pegaga TGGACGCGCGGGCCTAATCATTCAGATCCATCAGAGACTCTAGTGCCACA 50 Norovirus Kangkung TGGACGCGTGGGCCTAATCATTCAGATCCATCAGAGACTCTAGTGCCACA 50 Norovirus Tauge TGGACGCGTGGGCCTAACCATTCAGATCCATCAGAGACTCTAGTGCCACA 50 Norovirus Kesum TGGACGCGTGGGCCCAATCATTCAGATCCATCAGAGACTCTAGTGCCACA 50 ************************************************** Norovirus Pegaga CACTCAAAGAAAA-TACAGTTGATTTCACTTCTAGGGGAAGCTTCACTCC 99 Norovirus Kangkung CACTCAAAGAAAAATACAGTTGATTTCACTTCTAGGGGAAGCTTCACTCC 100 Norovirus Tauge CACTCAAAGAAAAATACAGTTGATTTCACTTCTAGGGGAAGCTTCACTCC 100 Norovirus Kesum CACTCAAAGAAAAATACAGTTGATTTCACTTCTAGGGGAAGCTTCACTCC 100 ************************************************* Norovirus Pegaga ATGGTGAGAAATTTTACAGAAAGATTTCCAGCAAGGTCATACATGAAATC 149 Norovirus Kangkung ATGGTGAGAAATTTTACAGAAAGATTTCCAGCAAGGTCATACATGAAATC 150 Norovirus Tauge ATGGTGAGAAATTTTACAGAAAGATTTCCAGCAAGGTCATACATGAAATC 150 Norovirus Kesum ATGGTGAGAAATTTTACAGAAAGATTTCCAGCAAGGTCATACATGAAATC 150 ************************************************** Norovirus Pegaga AAGACTGGTGGATTGGAAATGTATGTCCCAGGATGGCAGGCCATGTTCCG 199 Norovirus Kangkung AAGACTGGTGGATTGGAAATGTATGTCCCAGGATGGCAGGCCATGTTCCG 200 Norovirus Tauge AAGACTGGTGGATTGGAAATGTATGTCCCAGGATGGCAGGCCATGTTCCG 200 Norovirus Kesum AAGACTGGTGGATTGGAAATGTATGTCCCAGGATGGCAGGCCATGTTCCG 200 ************************************************** Norovirus Pegaga TTGG-ATGCGGTTAT----- 213 Norovirus Kangkung TTGG-ATGCGGTTATCTT-- 217 Norovirus Tauge TTGG-ATGCGGTTC------ 213 Norovirus Kesum TTGG-ATGCGATCT------ 213 **************************
GI Phylogenetic analysis of Norovirus strains based on RNA-dependent RNA-polymerase region (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 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 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).
ACKNOWLEDGEMENTS Food Quality and Safety Division Financial Support National Public Health Laboratory, Sungai Buloh University Putra Malaysia