Virus Contamination in Produce Y. Carol Shieh, Ph.D., carol.shieh@fda.hhs.gov FDA Moffett Center National Center for Food Safety and Technology Summit-Argo, IL

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

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

Pathogen-known Foodborne Illnesses Estimated by CDC BACTERIA Campylobacter spp. 1,963,141 14.2% Salmonella. nontyphoidal 1,341,873 9.7% Clostridium perfringens 248,520 1.8% Subtotal 4,175,565 30.2% VIRUSES Noroviruses 9,200,000 66.6% Rotavirus 39,000 0.3% Astrovirus 39,000 0.3% Hepatitis A virus 4,170 0.0% Subtotal 9,282,170 67.2% Grand Total 13,814,924 100% Emerging Infectious Diseases, 1999, 5: 607

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.

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.

Different Food Matrices Implicated in US Norovirus Outbreaks Emerging Infectious Diseases, 2005, 11 (1): 95 1998-2000 Outbreak Number

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

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.

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.

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.

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

Cellular Infectivity of HAV 04-22-08

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

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

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

Fluorogenic RT-PCR of HAV CT: HAV (U): 28.6 5 x 102 30.3 1 x 102 32.5 5 x 101 34.1 1 x 101 37.0 5 x 100 38.0 1 x 100 nd 5 x 10-1 39.6 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.

Standard Curve for HAV Unit Estimation Log (HAV units) = -0.281 x ( CT) + 10.67; r2 = 0.98 2-21-2007

PCR Unit vs. Infectivity of 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 HAV Strains from Food and Patients Nucleotide Sequence Dendogram

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.

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.

Viral Pathogens Handled in BSL-2 Hood

Virus inoculation and drying Performed in BSL-2 Hood

Virus Elution from Baby Spinach and Green Onions

Virus Elution Assisted by Shaking

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

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.

Evaluate Different Eluents to Recover HAV from Green Onions Eluent CT Estimated Recovery 1. HAV control 33.2±0.3 100% 2. PBS, pH 7.5 33.7±1.7 73% (cell culture grade) 3. Butterfield 34.1±0.6 56% phosphate buffer, pH 7 4. Water 34.5±0.4 43%

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 7.5 52% 36% b. BE (B Co.), pH 8 47% 38% c. BE (D Co.), pH 7.5 50% 34% d. BE (D Co.), pH 8 50% 33%

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

Additional Challenge: PCR-inhibitors naturally occurring in food & water Sample Water O62 CT RNA (µl) (cycle no.) PV, positive 0 35.8 PV + O62 2 36.9 PV + O62 5 39.1 PV + O62 8 or 10 nd Neg control 0 nd

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 98-100 ºC, 10 min (4) 95ºC, 2 min, to activate Platinum Taq and followed by 20 min RT

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.

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