1. Virus Contamination in Produce
Y. Carol Shieh, Ph.D.,
FDA Moffett Center
National Center for Food Safety and Technology
Summit-Argo, IL

2. Virus Contamination in Produce
An update on virus contamination in produce
A laboratory demonstration on the detection of hepatitis A virus in spinach & green onions using fluorogenic RT-PCR, real time RT-PCR

3. US Foodborne Diseases Estimated by CDC
76 million illnesses/year
325,000 hospitalizations/year
5,000 deaths/year
14 million illnesses with pathogens known
Emerging Infectious Diseases, 1999, 5: 607

4. Pathogen-known Foodborne Illnesses Estimated by CDC
BACTERIA
Campylobacter spp. 1,963, %
Salmonella. nontyphoidal 1,341, %
Clostridium perfringens , %
Subtotal ,175, %
VIRUSES
Noroviruses 9,200, %
Rotavirus , %
Astrovirus , %
Hepatitis A virus , %
Subtotal ,282, %
Grand Total 13,814, %
Emerging Infectious Diseases, 1999, 5: 607

5. Foodborne Viruses Norovirus (NoV): an RNA virus of Caliciviridae
NoV is responsible for 50% of US foodborne outbreaks of gastroenteritis.
Immunity is not long-lasting after infection.
Hepatitis A virus: an RNA virus of Picornaviridae
Immunity after infection is lifelong.
The median incubation period is 28 days.

6. Characteristics of Viral Contamination in Food
Viruses are transmitted primarily by fecal-oral route.
Human enteric viruses do not replicate but persist in food or in the environment.
Produce, shellfish, and ready-to-eat food have been major vehicles for foodborne viral diseases.

7. Different Food Matrices Implicated in US Norovirus Outbreaks
Emerging Infectious Diseases, 2005, 11 (1): 95
Outbreak Number

8. Virus Entries into Food
Harvest Field
Before & during harvest
During food processing
During final preparation
Human Consumption

9. Probable Causes of Produce-implicated Outbreaks
In 1990, in U.S., two HAV outbreaks were linked to similar lots of frozen strawberries. Identical HAV was found in the outbreak patients.
Viral contamination occurred prior to the product distribution.

10. Probable Causes
Three thousand cases of gastroenteritis occurring in a 1991 outbreak in Australia were attributed to norovirus-contaminated orange juice from a manufacturer.
The illnesses diminished upon the product withdrawal.
An investigation concluded
faulty plumbing in the processing plant.

11. Green onions-implicated HAV Outbreak, 2003
MMWR 52(47):1155
Approximately 555 hepatitis A patients were identified, including 13 workers and 75 out-of-state diners at a restaurant.
Green onions were grown in Mexico, shipped to the restaurant, stored at refrigeration temp., and prepared within a few days.

12. Techniques for Studying Virus Contamination in Produce
Molecular assays are rapid and sensitive.
Infectivity assays provide accuracy in assessing risks.

13. Cellular Infectivity of HAV

14. Plaque Assay
30 μl
100 μl
200 μl
35 mm in diameter well

15. Polymerase Chain Reaction (PCR) for Pathogen Detection
Heat-stable Taq polymerase duplicates target DNA.
PCR profile consists of target DNA denaturation, primer annealing, and nucleotide extension.
PCR exponentially amplifies target DNA. 1 2 3 4

16. Fluorogenic Reverse Transcription (RT)-PCR for Virus Detection
Primer 1
Primer 2
probe labeled with a reporter at 5'-end
& a quencher at 3'-end

17. Fluorogenic RT-PCR of HAV
CT: HAV (U):
x 102
x 102
x 101
x 101
x 100
x 100
nd 5 x 10-1
x 10-1
HAV primers and probe by Jothikumar et al.
AEM 2005 vol. 71(6):3359
Minor adjustment was made for PCR in Opticon.

18. Standard Curve for HAV Unit Estimation
Log (HAV units) = x ( CT) ; r2 = 0.98

19. PCR Unit vs. Infectivity of HAV
One unit of HAV HM-175 per reaction detectable by fluorogenic RT-PCR was PFU.
The ratio of PCR units to infectivity was found to be 45:1.
11/19/2007

20. Molecular Techniques Used in an Outbreak Investigation
In a 2005 multi-state hepatitis A outbreak,
Thirty-nine patients of hepatitis A were reported in AL, FL, SC and TN.
All patients had consumed oysters harvested from specific areas.
Journal of Food Protection, 2007, vol. 70 :145

21. Are Molecular Techniques Useful?
HAV was detected in oysters using FDA GCSL virus extraction protocol and fluorogenic RT-PCR.
The HAV strain from oysters was identified by nucleotide-sequencing, and comparing to the HAV strain of 28 patient serum specimens.

22. HAV Amplicons from Outbreak Oysters

23. Genetic Relatedness of HAV Strains from Food and Patients
Nucleotide Sequence Dendogram

24. Use of Molecular Techniques Allowed the Following Conclusion
A common source of viral contamination occurred prior to product distribution.
Contaminated products were confirmed as the transmission vehicle.
The highest incidence of HAV contamination was estimated to be one in every 11 oysters examined.

25. Rapid Assays for Viruses Used in Current Projects at Moffett Center
One step fluorogenic RT-PCR currently is used to evaluate HAV recoveries from green onions and baby spinach.
Norovirus cross-contamination during foodservice procedure will be studied.

26. Viral Pathogens Handled in BSL-2 Hood

27. Virus inoculation and drying Performed in BSL-2 Hood

28. Virus Elution from Baby Spinach and Green Onions

29. Virus Elution Assisted by Shaking

30. Virus Elution and Extraction Procedure
1. Inoculate 10 μl of virus stock
2. Air-dry 30 min in BSL-2 hood
3. Shake the sample in 10 ml eluent
at 145 rpm, 15 min, 20ºC
4. Concentrate viruses if necessary, e.g.
PEG-precipitation, use of Centricon
5. Extract RNAs if necessary

31. HAV in Eluates Detected by RT-PCR
HAV was eluted and detected by fluorogenic RT-PCR.
HAV units in eluates were estimated via a standard curve with specific CT value.
Log (HAV units) = a x CT +b.
HAV recovery was calculated by comparing to positive controls.

32. Evaluate Different Eluents to Recover HAV from Green Onions
Eluent CT Estimated
Recovery
1. HAV control 33.2± %
2. PBS, pH ± %
(cell culture grade)
3. Butterfield ± %
phosphate buffer, pH 7
4. Water ± %

33. HAV Eluted from Baby Spinach
Eluent Estimated Bio-recovery
recovery (PFU)
(CT)
1. PBS/2% serum 60% 39%
2. beef extract (BE)
a. BE (B Co.), pH % 36%
b. BE (B Co.), pH % 38%
c. BE (D Co.), pH % 34%
d. BE (D Co.), pH % 33%

34. Compare Estimated Recoveries to Bio-recoveries
HAV Recovered from BS
HAV Recovery

35. Additional Challenge:
PCR-inhibitors naturally occurring in food & water
Sample Water O CT
RNA (µl) (cycle no.)
PV, positive
PV + O
PV + O
PV + O or nd
Neg control nd

36. One Step RT-PCR Protocol to be Used in the Workshop Lab Session
The protocol was modified from
Jothikumar’s HAV TaqMan assay:
(1) Invitrogen SuperScript III Platinum
One-step Quantitative RT-PCR kit
(2) A volume of 25 μl/rx in Smart Cycler
(3) An initial step to heat-rupture virions using ºC, 10 min
(4) 95ºC, 2 min, to activate Platinum Taq and followed by 20 min RT

37. Summary
Rapid detection of foodborne viruses can be facilitated by molecular techniques.
Levels of viruses detected by rapid molecular assays possibly differ from that of infectivity assays.
Overcoming inhibitors is essential for a successful rapid detection of viruses in food matrix.

38. National Center for Food Safety and Technology
Acknowledgement
David Laird
Karl Reineke
Diane Stewart
Lou Tortorello
Rich McDonald
Martin Cole
FDA Moffett Center
National Center for Food Safety and Technology
Summit-Argo, IL 60501