National Institutefor Public Healthand the Environment Persistence and Inactivation of

Norovirus in Fresh Produce Chains

Katharina Verhaelen

National Institutefor Public Healthand 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

40%

18%

14%

8%

6%

3%4%

3% 3% Norovirus

Salmonella

Other

E. coli

Clostridium

Staphylococcus

Shigella

Bacillus

Staphylococcus

DeWaal and Buhiya, 2007

National Institutefor Public Healthand 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

National Institutefor Public Healthand the Environment

Life of a berry

ProcessedFresh

Dried Puree Juice Frozen

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

National Institutefor Public Healthand the Environment

Life of a berry

ProcessedFresh

Dried Puree Juice Frozen

Conditions/Preservation Measures

• Pasteurisation:

• Scenario a) 88°C, 2min; Scenario b1) 65°C, 30s or b2)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: b1) 1.8 log b2) 2.8 log

National Institutefor Public Healthand the Environment

Life of a berry

ProcessedFresh

Dried Puree Juice Frozen

Conditions/Preservation Measures

• Freezing

• Literature data: (Butot, 2008)

• FCV log (TCID50) 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

National Institutefor Public Healthand the Environment

Life of a berry

ProcessedFresh

Dried Puree Juice Frozen

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

National Institutefor Public Healthand the Environment

Life of a berry

ProcessedFresh

Dried Puree Juice Frozen

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

National Institutefor Public Healthand 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

National Institutefor Public Healthand the Environment

Approach

Controls

Process control:

• Mengovirus

Plaque assay

• hAdV: Virobathe protocol

• MNV: Wobus, 2004

Cen

trifu

gatio

n10

000

rpm

Cen

trifu

gatio

n10

000

rpm

Spike food with virus

Virus inactivation

Elution of virus particles from food surfaceusing 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 PCR Plaque assay

Cen

trifu

gatio

n10

000

rpm

Sam

ple

taki

ngdu

ring

time

National Institutefor Public Healthand 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 T0 control• Cp (hAdV): 31-32• Cp (MNV-1) 10x dilution: 33-34

• Infectivity data not yet available difficulties in culturing RAW 264.7 cells

National Institutefor Public Healthand the Environment

Conditions

Mimic storage conditions of fresh and frozen raspberries/strawberries:

Temperature profile:

Harvest Distribution Point of sale

10 °C

RT

4 °C

7 d

Point of saleHarvest Distribution

10 °C

RT

4 °C

14 d

National Institutefor Public Healthand 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

National Institutefor Public Healthand 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)

National Institutefor Public Healthand 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!