Virus Contamination Virus Contamination in Producein Produce

FDA Moffett CenterFDA Moffett CenterNational Center for Food Safety and National Center for Food Safety and

TechnologyTechnologySummit-Argo, IL Summit-Argo, IL

Y. Carol Shieh, Ph.D., carol.shieh@fda.hhs.gov

Virus Contamination Virus Contamination in Producein 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

US Foodborne Diseases US Foodborne Diseases Estimated by CDCEstimated by CDC

76 million illnesses/year76 million illnesses/year 325,000 hospitalizations/year325,000 hospitalizations/year

5,000 deaths/year5,000 deaths/year

14 million illnesses with pathogens 14 million illnesses with pathogens knownknown

Emerging Infectious Diseases, Emerging Infectious Diseases, 1999, 5: 6071999, 5: 607

Pathogen-known Pathogen-known Foodborne Illnesses Foodborne Illnesses Estimated by CDCEstimated by CDC

BACTERIA BACTERIA Campylobacter spp. Campylobacter spp. 1,963,141 1,963,141

14.2%14.2% Salmonella. nontyphoidal 1,341,873 Salmonella. nontyphoidal 1,341,873 9.7%9.7%

Clostridium perfringens Clostridium perfringens 248,520 248,520 1.8%1.8%

Subtotal Subtotal 4,175,565 4,175,565 30.2%30.2%

VIRUSES VIRUSES NorovirusesNoroviruses 9,200,000 9,200,000 66.6% 66.6% Rotavirus Rotavirus 39,000 39,000

0.3% 0.3% Astrovirus 39,000 Astrovirus 39,000 0.3%0.3% Hepatitis A virus 4,170 Hepatitis A virus 4,170 0.0%0.0%

SubtotalSubtotal 9,282,170 9,282,170 6 67.2%7.2%

Grand TotalGrand Total 13,814,92413,814,924 100% 100%

Emerging Infectious Diseases, Emerging Infectious Diseases, 1999, 5: 6071999, 5: 607

Foodborne VirusesFoodborne Viruses

Norovirus (NoV): an RNA virus of Norovirus (NoV): an RNA virus of Caliciviridae Caliciviridae NoV is responsible for NoV is responsible for 50% of US 50% of US foodborne outbreaks of gastroenteritis.foodborne outbreaks of gastroenteritis. Immunity is not long-lasting after Immunity is not long-lasting after infection.infection.

Hepatitis A virus: an RNA virus of Hepatitis A virus: an RNA virus of PicornaviridaePicornaviridae Immunity after infection is lifelong.Immunity after infection is lifelong. The median incubation period is The median incubation period is 28 days.28 days.

Characteristics of Characteristics of Viral Contamination in Viral Contamination in

FoodFood

Viruses are transmitted primarily Viruses are transmitted primarily by fecal-oral route. by fecal-oral route.

Human enteric viruses do not Human enteric viruses do not replicate but persist in food or in the replicate but persist in food or in the environment.environment.

Produce, shellfish, and ready-to-eat Produce, shellfish, and ready-to-eat food have been major vehicles for food have been major vehicles for foodborne viral diseases.foodborne viral diseases.

Different Food Matrices Different Food Matrices Implicated in Implicated in

US Norovirus OutbreaksUS Norovirus Outbreaks

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Emerging Infectious Diseases, 2005, 11 (1): 95

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Virus Entries into Virus Entries into FoodFood

Harvest Field

Before & during harvest

During food processing

During final preparation

Human Consumption

Probable Causes ofProbable Causes ofProduce-implicated Produce-implicated

OutbreaksOutbreaks

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.

Probable CausesProbable 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.

Green onions-Green onions-implicated implicated

HAV Outbreak, 2003HAV Outbreak, 2003

MMWR 52(47):1155MMWR 52(47):1155

Approximately 555 hepatitis A Approximately 555 hepatitis A patients were identified, including patients were identified, including 13 workers and 75 out-of-state 13 workers and 75 out-of-state diners at a restaurant. diners at a restaurant.

Green onions were grown in Green onions were grown in Mexico, shipped to the restaurant, Mexico, shipped to the restaurant, stored at refrigeration temp., and stored at refrigeration temp., and prepared within a few days. prepared within a few days.

Techniques for Studying Techniques for Studying Virus Contamination in Virus Contamination in

ProduceProduce

Molecular assays are rapid and Molecular assays are rapid and sensitive.sensitive.

Infectivity assays provide accuracy Infectivity assays provide accuracy in assessing risks. in assessing risks.

Cellular Infectivity Cellular Infectivity of HAVof HAV

04-22-08

Plaque AssayPlaque Assay

35 mm in diameter well

30 30 μμll 100100 μμll 200 200 μμll

Polymerase Chain Reaction (PCR) for Pathogen DetectionDetection

Heat-stable Taq polymerase Heat-stable Taq polymerase duplicates target DNA.duplicates target DNA. PCR profile consists of target DNA PCR profile consists of target DNA denaturation, primer annealing, and denaturation, primer annealing, and nucleotide extension. nucleotide extension. PCR exponentially amplifies target PCR exponentially amplifies target DNA.DNA.

11 22 33 44

Fluorogenic Reverse Fluorogenic Reverse Transcription (RT)-PCR Transcription (RT)-PCR

for Virus Detectionfor Virus Detection

Fluorogenic RT-PCR Fluorogenic RT-PCR of HAVof HAV

CT: CT: HAV (U):HAV (U):28.628.6 5 x 105 x 1022

30.330.3 1 x 101 x 1022

32.532.5 5 x 105 x 1011

34.134.1 1 x 101 x 1011

37.037.0 5 x 105 x 1000

38.038.0 1 x 101 x 1000

ndnd 5 x 105 x 10-1-1

39.639.6 1 x 101 x 10-1-1

HAV primers and probe by Jothikumar et al. AEM 2005 vol. 71(6):3359

Minor adjustment was made for PCR in Opticon.

Standard Curve for Standard Curve for HAV Unit EstimationHAV Unit Estimation

Log (HAV units) = -0.281 x (Log (HAV units) = -0.281 x ( CT) + 10.67; rCT) + 10.67; r2 2 = = 0.980.98

2-21-2007

PCR Unit vs. Infectivity PCR Unit vs. Infectivity of HAVof HAV

One unit of HAV HM-175 per reaction detectable by fluorogenic RT-PCR was 0.022 PFU.

The ratio of PCR units to infectivity was found to be 45:1. 11/19/2007

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

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.

HAV Amplicons from Outbreak Oysters

Genetic Relatedness of Genetic Relatedness of HAV Strains from HAV Strains from Food and PatientsFood and Patients

Nucleotide Sequence Dendogram

Use of Molecular Techniques Allowed the Following

Conclusion1. A common source of viral contamination

occurred prior to product distribution.

2. Contaminated products were confirmed as the transmission vehicle.

3. The highest incidence of HAV contamination was estimated to be one in every 11 oysters examined.

Rapid Assays for Viruses Rapid Assays for Viruses Used in Current Projects Used in Current Projects

at Moffett Centerat Moffett Center One step fluorogenic RT-PCR One step fluorogenic RT-PCR currently is used to evaluate HAV currently is used to evaluate HAV recoveries from green onions and recoveries from green onions and baby spinach.baby spinach.

Norovirus cross-contamination Norovirus cross-contamination during foodservice procedure will be during foodservice procedure will be studied.studied.

Viral Pathogens Handled Viral Pathogens Handled in BSL-2 Hood in BSL-2 Hood

Virus inoculation and Virus inoculation and drying drying

Performed in BSL-2 HoodPerformed in BSL-2 Hood

Virus Elution from Baby Virus Elution from Baby Spinach and Green Spinach and Green

OnionsOnions

Virus Elution Virus Elution Assisted by ShakingAssisted by Shaking

Virus Elution and Virus Elution and Extraction ProcedureExtraction Procedure

1. 1. Inoculate 10 Inoculate 10 μμl of virus stockl of virus stock

2.2. Air-dry 30 min in BSL-2 hood Air-dry 30 min in BSL-2 hood

3.3. Shake the sample in 10 ml eluent Shake the sample in 10 ml eluent at 145 rpm, 15 min, 20ºCat 145 rpm, 15 min, 20ºC

4.4. Concentrate viruses if necessary, Concentrate viruses if necessary, e.g. e.g.

PEG-precipitation, use of PEG-precipitation, use of CentriconCentricon

5.5. Extract RNAs if necessary Extract RNAs if necessary

HAV in Eluates HAV in Eluates Detected by RT-PCRDetected by RT-PCR

HAV was eluted and detected by HAV was eluted and detected by fluorogenic RT-PCR.fluorogenic RT-PCR.

HAV units in eluates were HAV units in eluates were estimated via a standard curve with estimated via a standard curve with specific CT value.specific CT value.Log (HAV units) = a Log (HAV units) = a x x CT CT ++b. b.

HAV recovery was calculated by HAV recovery was calculated by comparing to positive controls.comparing to positive controls.

Evaluate Different Evaluate Different Eluents Eluents

to Recover HAVto Recover HAVfrom Green Onionsfrom Green Onions

EluentEluent CTCT EstimatedEstimated

RecoveryRecovery

1.1. HAV control HAV control 33.233.2±0.3±0.3 100% 100%

2.2. PBS, pH 7.5 PBS, pH 7.5 33.733.7±1.7±1.7 73%73%

(cell culture grade)(cell culture grade)

3.3. Butterfield Butterfield 34.134.1±0.6 ±0.6 56%56%

phosphate buffer, pH 7 phosphate buffer, pH 7

4.4. Water Water 34.534.5±0.4±0.4 43%43%

HAV Eluted from HAV Eluted from Baby Spinach Baby Spinach

EluentEluent Estimated Estimated Bio- Bio-recoveryrecovery

recoveryrecovery (PFU)(PFU)

(CT)(CT)1. PBS/2% serum1. PBS/2% serum 60% 60% 39%39%2. beef extract (BE) 2. beef extract (BE) a. BE (B Co.), pH 7.5a. BE (B Co.), pH 7.5 52% 52%

36% 36% b. BE (B Co.), pH 8b. BE (B Co.), pH 8 47% 47% 38% 38% c. BE (D Co.), pH 7.5c. BE (D Co.), pH 7.5 50% 50%

34% 34% d. BE (D Co.), pH 8d. BE (D Co.), pH 8 50% 50% 33%33%

Compare Estimated Compare Estimated Recoveries to Bio-Recoveries to Bio-

recoveriesrecoveries

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Recovery

HA

V R

ecovery

HAV Recovered from BS

Sample Sample Water O62 Water O62 CT CT RNA RNA (µl)(µl) (cycle no.) (cycle no.)

PV, positivePV, positive 0 0 35.835.8

PV + O62PV + O62 2 2 36.936.9

PV + O62PV + O62 5 5 39.139.1

PV + O62PV + O62 8 or 10 8 or 10 nd nd

Neg controlNeg control 0 0 nd nd

Additional Challenge: Additional Challenge: PCR-inhibitors naturally PCR-inhibitors naturally

occurring in food & wateroccurring in food & water

One Step RT-PCR Protocol to be One Step RT-PCR Protocol to be

Used in the Workshop Lab Used in the Workshop Lab SessionSession

The protocol was modified fromJothikumar’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 98-100 ºC, 10 min

(4) 95ºC, 2 min, to activate Platinum Taq and followed by 20 min RT

Summary 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.

AcknowledgementAcknowledgement

David LairdDavid LairdKarl Reineke Karl Reineke Diane StewartDiane Stewart

Lou TortorelloLou TortorelloRich McDonaldRich McDonaldMartin ColeMartin Cole

FDA Moffett CenterFDA Moffett CenterNational Center for Food Safety and TechnologyNational Center for Food Safety and Technology

Summit-Argo, IL 60501Summit-Argo, IL 60501

David LairdDavid LairdKarl Reineke Karl Reineke Diane StewartDiane Stewart

Lou TortorelloLou TortorelloRich McDonaldRich McDonaldMartin ColeMartin Cole

FDA Moffett CenterFDA Moffett CenterNational Center for Food Safety and TechnologyNational Center for Food Safety and Technology

Summit-Argo, IL 60501Summit-Argo, IL 60501