1. National Institute for Public Health and the Environment Persistence and Inactivation of Norovirus in Fresh Produce Chains Katharina Verhaelen

2. National Institute for Public Health and the Environment Introduction  Human Noroviruses (hNoV) are the leading cause of food- borne viral gastroenteritis worldwide  Number of reported outbreaks attributed to fresh produce increasing  hNoV is the most important cause of fresh produce associated outbreaks DeWaal and Buhiya, 2007

3. National Institute for Public Health and the Environment Work package 6 Objectives 1.To determine the persistence of NoV under conditions encountered in food supply chains 2.To asses the efficacy of current and novel intervention measures in inactivating NoV 3.To verify the disinfectant efficacy of interventions at pilot plant scale and in field experiments for hNoV  Gathered quantitative data on NoV reduction will feed back into QVRA

4. National Institute for Public Health and the Environment Life of a berry ProcessedFresh DriedPureeJuiceFrozen Conditions/Preservation Measures Freeze drying: Freezing, Heat + reduced pressure  sublimation of ice Literature data: (Butot, 2009) Raspberries/Strawberries: reduction hNoV GI ~0.7 log, hNoV GII ~1.3 log

5. National Institute for Public Health and the Environment Life of a berry ProcessedFresh DriedPureeJuiceFrozen Conditions/Preservation Measures Pasteurisation: Scenario a) 88°C, 2min; Scenario b 1 ) 65°C, 30s or b 2 )75 °C, 15s Literature data: (Baert, 2008) MNV-1 log (PFU) reduction in buffer at 80°C, 2.5min : a) > 6 log MNV-1 log (PFU) reduction in rasp. puree: b 1 ) 1.8 log b 2 ) 2.8 log

6. National Institute for Public Health and the Environment Life of a berry ProcessedFresh DriedPureeJuiceFrozen Conditions/Preservation Measures Freezing Literature data: (Butot, 2008) FCV log (TCID 50 ) reduction, 2 days: raspb. 1.1 log; strawb. 2.7 log hNoV GI/GII (PCRU) reduction, 2 days, raspberry: no reduction hNoV GI/GII (PCRU) reduction, 2 days, strawberry: < 0.4 log

7. National Institute for Public Health and the Environment Life of a berry ProcessedFresh DriedPureeJuiceFrozen Conditions/Preservation Measures Chilling Literature data: (Mattison, 2007) FCV reduction (PFU), strawberries, 6 days, 4°C: > 3.5 log reduction No data on hNoV/MNV-1 persistence on raspberries/strawberries

8. National Institute for Public Health and the Environment Life of a berry ProcessedFresh DriedPureeJuiceFrozen Conclusion  If data available often inconclusive/incomplete  Study persistence on fresh and frozen berries  Highest risk (short shelf life, conditions preserve virus infectivity)  Highest consumption

9. National Institute for Public Health and the Environment Viruses under Study hNoV GI.4 & GII.4  target pathogen Murine norovirus (MNV-1)  surrogate to study hNoV infectivity Human adenovirus (hAdV)  indicator for hNoV Low and high virus titer

10. National Institute for Public Health and the Environment Approach Controls Process control: Mengovirus Plaque assay hAdV: Virobathe protocol MNV: Wobus, 2004 Centrifugation 10000 rpm Spike food with virus Virus inactivation Elution of virus particles from food surface using TGBE buffer pH 9.5 Chloroform:Butanol extraction Precipitation of virus particles using polyethylene glycol pH 7.2 Filtration Nucleic acid extraction using magnetic silica Quantitative PCRPlaque assay Centrifugation 10000 rpm Sample taking during time

11. National Institute for Public Health and the Environment Pre-Experiment Aim: Determination of experimental set up Initial virus concentration  low recovery Sampling intervals Testing the whole procedure Approach: 10g raspberry spiked with 5000 PFU of MNV-1 and hAdV Duplicates Storage of raspberries at 4°C for 1 week and at room temperature for 2 days Preliminary results: No decrease in Cp values for hAdV and MNV-1 after 7 days of storage at 4°C and after 2 days at RT compared to T 0 control Cp (hAdV): 31-32 Cp (MNV-1) 10x dilution: 33-34 Infectivity data not yet available  difficulties in culturing RAW 264.7 cells

12. National Institute for Public Health and the Environment Conditions  Mimic storage conditions of fresh and frozen raspberries/strawberries: Temperature profile: HarvestDistributionPoint of sale 10 °C RT 4 °C 7 d Point of saleHarvestDistribution 10 °C RT 4 °C 14 d

13. National Institute for Public Health and the Environment Expected Outcome Knowledge on persistence of hNoV on soft berries under commonly applied storage conditions will provide data for:  Setting health targets  Assessing public health risk  Redesigning food processing practices hAdV useful indicator of hNoV contamination of food commodities in respect to its persistence Suitability of MNV-1 as a surrogate of hNoV removal Differences in persistence of hNoV GI and GII Impact of food matrix on hNoV persistence

14. National Institute for Public Health and the Environment Outlook for this year 1.Persistence of hNoV in agrochemicals  Application of agrochemicals possible introduction source for hNoV on fresh produce? In the raspberry production insecticides are applied prior to mechanical harvesting to avoid contamination of produce Direct contact with produce 2.Inactivation of hNoV in raspberry puree by high pressure processing on industrial scale (ITACyL) 3.Efficacy of lettuce washing with and without inclusion of sanitizers in inactivating hNoV on pilot plant scale (ITACyL)

15. National Institute for Public Health and the Environment Acknowledgement: Ana Maria de Roda Husman Saskia Rutjes Martijn Bouwknegt Froukje Lodder Mohammed Alsady Marjolijn Schalk Willemijn Lodder Erwin Duizer & Era Tuladhar Arie Havelaar Frans van Knapen Thanks for your attention!