Noroviruses in Shellfish and Other Foods: Challenges of the 21st Century
By Gary P. Richards, Ph.D., and David H. Kingsley, Ph.D.
If you were unfortunate enough to have a bout of norovirus illness during your lifetime, you probably still remember it. Nothing is more disheartening than to develop norovirus illness while traveling on that well-deserved vacation or after a wedding reception or celebratory meal at your favorite restaurant. The fact is, noroviruses are the principle cause of foodborne illness. In the United States alone, there are an estimated 5.5 million cases annually. Although other enteric viruses contribute to foodborne illness, noroviruses are by far the most prevalent. Noroviruses are transmitted by ingesting the pathogen, through contaminated food, water or person-to-person contact. They are highly contagious and found in high numbers in both feces and vomit.
Norovirus particles can cause illness even in relatively small numbers, estimated at perhaps as few as ten. Symptoms often include projectile vomiting and explosive diarrhea, usually at the same time, making this illness most unpleasant. Combating noroviruses in the food supply chain takes an all-hands-on-deck approach involving growers, harvesters, transporters, processors, food handlers and of course, consumers. Some practical measures to reduce noroviruses in foods involve pre- and postharvest interventions, product testing and targeted research.
Poor Hygienic Practices
One of the most common means for food contamination is poor personal hygiene, which leads to hand contamination and subsequent transfer of viruses to foods simply by handling. Foods commonly implicated in enteric virus illness include fruits and vegetables, deli meats, bakery products, ready-to-eat foods and molluscan shellfish. Foods handled by ill workers or persons with unsanitary hands are frequently the source of contamination, at harvest, during transport, at the grocery store or during food preparation and serving. Fruits, vegetables and berries may be contaminated in the field by the harvesters’ hands, but also by polluted irrigation water or sewage-contaminated drainage ditches or septic systems in the vicinity of the farms. Maintaining clean dishes and silverware is also important in reducing illnesses. Hand washing with soap and water prior to harvest, transport and preparation of foods is essential to reducing norovirus illnesses in the general population. If gloves are used to handle foods, they should be maintained in a sanitary manner. Noroviruses are generally resistant to alcohol-based products, including hand sanitizers, so thorough washing is very important.
Environmental Contamination of Shellfish
Unlike most food products, in which handling is often the source of contamination, bivalve molluscan shellfish (oysters, clams, mussels and cockles) are most commonly contaminated by fecally polluted water in the harvest area. These shellfish feed by filtering out particles in the water through their gills and diverting the particles to their mouths and digestive tracts. Bivalves can bioconcentrate viruses within their edible tissues to many times the levels in the surrounding water. This makes raw shellfish susceptible to high levels of norovirus contamination when the water is polluted. To minimize illnesses, regulators in the U.S. monitor shellfish growing waters for signs of fecal pollution using fecal coliform (bacterial) standards. These bacteria are not the best indicators for virus pollution, because bacteria and viruses have different tolerances to ever-changing environmental conditions. Nevertheless, the detection of bacterial contamination of fecal origin signifies the likelihood that norovirus and other enteric viruses are also present. The converse is not true; that is, water that tests clean for fecal coliform bacteria may still contain viruses that are more persistent than bacteria. Shellfish from such waters could still pose a threat to the consumer. Other factors that contribute to shellfish contamination include storm water runoff, particularly in areas with septic tanks, faulty sewage treatment plants and the illicit dumping of boat wastes into harvesting areas. Dumping of boat wastes and vomiting overboard have been associated with specific outbreaks.[3, 4] Harvesting only from areas approved by state regulators will reduce the threat of illness but cannot guarantee safety. Other factors, like storing shellfish in a sanitary manner, chilling them with “clean” ice and handling the shell stock and shucked products under hygienic conditions, are necessary precautions to enhance shellfish safety.
Washing. Since honest efforts can go only so far in providing safe foods, postharvest processing interventions are often necessary to enhance food safety. The outer surfaces of foods such as fruits and vegetables, leafy greens and berries should be washed with potable water to remove surface contamination to the greatest extent possible. Foods with porous or crinkly surfaces, like strawberries and curly leaf lettuce or spinach, are more difficult to wash and may require more effort to remove surface contamination. Several rinses of produce with copious amounts of water would be expected to lessen the number of viruses present. Even then, viruses may remain within pores, cracks or surface folds. Peeling vegetables will also remove surface contamination. Melons should be thoroughly washed before cutting to prevent the transfer of surface contamination to the inside of the fruit via the knife.
The outer shell of molluscan shellfish should be washed clean to remove mud and potential surface viruses, particularly if the shellfish are to be shucked and eaten raw. In a food processing environment, food contact surfaces should be washed with dilute bleach solution to inactivate (kill) potential noroviruses, although the effective concentrations and exposure times for bleach and other disinfectants against human noroviruses have not been defined.
Heating. Pasteurization and/or cooking are effective means to inactivate enteric viruses, but their effectiveness depends on the type of cooking (frying, steaming, baking, boiling) and the duration. Internal temperatures must be sufficient to inactivate viruses, although the times and temperatures required to inactivate human noroviruses have not been clearly defined. Cooking is effective and useful as a disinfection process for many foods, including vegetables. A short blanching may be sufficient to eliminate surface contaminants on fresh vegetables. For shellfish, thorough cooking can leave them overly chewy, so alternative processing strategies are needed.
Depuration. A popular method to cleanse shellfish of bacterial and other contaminants is known as controlled purification or depuration. Molluscan shellfish are placed in tanks of clean seawater and allowed to purge the contaminants from their systems, generally over a 3-day period. Depuration has been practiced for over 100 years and is effective in reducing bacterial contamination and sand, but is less efficient in reducing enteric viruses from shellfish. Enteric viruses apparently become sequestered in motile, phagocytic hemocytes (blood cells) of the shellfish. Motile, phagocytic hemocytes can travel back and forth from the digestive tract, through the epithelial cells surrounding the tract and into the shellfish’s connective tissues. Although these hemocytes have acidic interiors, enteric viruses can be highly acid tolerant. Laboratory studies have shown that hepatitis A virus was very acid tolerant and persisted for 21 days in oyster hemocytes, whereas other viruses were less tolerant. No studies of acid persistence of human norovirus have been performed, but judging from the number of norovirus illnesses caused by shellfish, it is suspected that noroviruses may also persist in hemocytes for extended periods. Preliminary testing suggests that many of the viruses within oysters may reside within hemocytes that migrate from the digestive tissues into the connective tissues of the shellfish. Consequently, shellfish depuration appears inadequate to purge viruses from shellfish.
High-Pressure Processing. High-pressure processing (HPP) is an alternative method to inactivate microbes in foods, including shellfish. HPP has been used to reduce vegetative bacteria in foods to enhance food safety and prolong shelf life. Foods like guacamole and fruit juices are often pressure treated. Pressure inactivates the spoilage enzymes in guacamole to preserve its green color, while HPP-treated fruit juices taste more like fresh-squeezed juices than heat-pasteurized products. Pressures of 250 MPa or higher are commonly used in the processing industry and kill most bacteria. A simple conversion for MPa is that 1 MPa = 145 psi of pressure, which is about five times what is normally used in a car tire. Therefore, 250 MPa is 36,250 psi. Pioneering work on enteric virus inactivation using HPP showed that hepatitis A virus and norovirus surrogates could be inactivated using moderate levels of pressure (450 MPa or less).[8, 9] In a collaboration between the U.S. Department of Agriculture (USDA), Emory University and Virginia Tech, it was determined that pressures greater than 400 MPa are required to inactivate norovirus particles in oysters using human volunteers. However, 400 MPa is higher than pressures used for commercial HPP treatments of shellfish.
Irradiation. Other postharvest processing methods include gamma and ultraviolet light irradiation. Gamma irradiation can be effective in eliminating some viruses, like hepatitis A virus, rotavirus, poliovirus and the norovirus surrogates feline calicivirus and canine norovirus; however, some studies reported that the levels required for inactivation negatively affected shellfish flavor. Ultraviolet light inactivates viruses on the surfaces of products, but is ineffective in the case of shellfish due to its inability to penetrate into the tissues where virus contamination generally resides. Procedures like salting or freezing and thawing appear relatively ineffective in reducing noroviruses.
Virus Analysis of Foods. Several methods have been developed to extract and test for total norovirus contamination (infectious and noninfectious virus particles) in foods; however, there are no internationally recognized standard methods to date. In an effort to develop standardized procedures, the European Committee for Standardization established a Technical Advisory Group for Viruses to develop and publish standard virus extraction and assay procedures for food surfaces, soft fruit and salad vegetables, bottled water and bivalve molluscan shellfish. After many years of work, their results should be available soon. Virus isolation from foods, either through a rinsing procedure or by extraction, must be followed by analysis of the viruses. In spite of improvements in our ability to extract viruses from foods, the analysis of rinses and extracts leaves much to be desired. Assay methods are almost exclusively based on reverse-transcription polymerase chain reaction (RT-PCR), a molecular-based procedure that amplifies viral RNA into complementary DNA (cDNA) copies. RT-PCR-based assays have a number of limitations. Perhaps the most significant is that they detect total virus presence (both infectious and noninfectious virus particles). Thus, viruses inactivated by chlorine, other disinfectants, sunlight, heat, high pressure, etc. can still test positive by RT-PCR. Other limitations of RT-PCR are that the assay is subject to laboratory contamination and is frequently inhibited by compounds in the extracts of shellfish or other foods. Various controls must be included in both virus extraction and assay to demonstrate the effectiveness of the extraction and the lack of inhibitors or contaminants in the assay. Such controls increase the time and complexity of the procedures but are necessary for accurate interpretation of the results.
Determining Norovirus Infectivity. For over 40 years, researchers have attempted to propagate noroviruses in culture. Unlike many human viruses, which can be assayed and quantified in cell culture, norovirus propagation has not been successful, in spite of some reports to the contrary. Plaque assays and cytopathogenicity assays are the basis for quantitative assessment for many viruses, but are ineffective for human noroviruses. Animal models are another way to monitor the infectivity of some viruses, but human noroviruses are incapable of replication in laboratory animals. Recent advances suggest it will soon be possible to separate inactive from potentially active norovirus using magnetic beads coated with molecules that mimic the cellular receptors to which noroviruses bind. It would then be possible to extract potentially infectious norovirus particles.[12, 13]
Inadequacies of Norovirus Surrogate Research
The quest for an assay to detect infectious noroviruses in food and water has been hampered by the inability to propagate human noroviruses in cell or tissue culture systems and the inability to infect common laboratory animals. Consequently, other viruses that can be assayed for infectivity are often used as norovirus surrogates. One of the earliest viruses to stake a claim as a norovirus surrogate was feline calicivirus, which produces easily quantified plaques in feline kidney cell culture. A more recent entry in the search for a surrogate is murine norovirus, which is genetically more similar to human norovirus than is feline calicivirus and produces plaques in a mouse macrophage cell line. To date, over 400 papers have been published on the use of feline calicivirus and murine norovirus to determine the effectiveness of chemical disinfectants and processing technologies on norovirus inactivation. Other norovirus surrogates have been proposed; however, it has become abundantly clear that none of the surrogates tested to date perfectly mimics human norovirus. In many cases, human noroviruses may be more persistent than the surrogates. Such variability in inactivation rates between the surrogate and the pathogen itself might have been anticipated, because different strains of even the same virus can have widely varying inactivation kinetics. For instance, different strains of feline calicivirus showed differences in their inactivation by chemicals, heat and pH,[15, 16] whereas different strains of hepatitis A virus showed substantial differences in inactivation by heat and HPP.17 Since human norovirus illnesses are caused by any of a wide variety of norovirus strains, it is unlikely that surrogate testing in itself will provide accurate data or data useful for the promulgation of regulations for the food industry. Currently, only human volunteer studies with the actual pathogens can definitively determine norovirus infectivity or the efficacy of sanitation interventions.
The Need for Clinical Trials
Clinical trials have been performed on human norovirus for decades, but the perceptions that they may be too risky or expensive have dissuaded some governments from funding such trials. In the United States, clinical trials are still funded by some agencies, and the information provided in respect to norovirus inactivation is essential to developing methods applicable to food processing. However, as a practical matter, the high cost and complexity of human trials limit the scope of this type of research. As previously mentioned, the results to date of surrogate-based studies are of limited value. Human clinical trials are needed to evaluate the effectiveness of disinfectants and processing technologies on human norovirus inactivation and to identify true norovirus surrogates. Only then will definitive information be available to properly evaluate the retention and disinfection of infectious noroviruses in foods.
Strategies to reduce human noroviruses in the food chain involve: (a) pre- and postharvest interventions to preclude noroviruses from food or food contact surfaces; (b) processing techniques to inactivate viruses on or within the products and (c) product analyses. Cooking, washing and peeling could have a significant effect on eliminating human noroviruses in some foods, but viruses tend to persist in molluscan shellfish. Efforts using norovirus surrogates identified to date have not resulted in new disinfectants or processing techniques, and have not significantly advanced food safety. Currently, human clinical trials remain the only method to conclusively show the effectiveness of processing techniques on virus persistence. Over the next decade, technological advancements may lead to simple, quantitative assays for human noroviruses. In the meantime, clinical trials will provide the best opportunity to identify processing interventions to reduce noroviruses in foods. The food industry, regulatory agencies and the public face many challenges in regard to norovirus contamination of the food supply, but with a concerted effort, obstacles that compromise food safety will be overcome.
Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA, which is an equal opportunity provider and employer.
Gary Richards, Ph.D., and David Kingsley, Ph.D., are research microbiologists at the USDA Agricultural Research Service’s Microbial Safety of Aquaculture Products Center of Excellence in Dover, DE. Their research involves the development of food safety and intervention technologies to reduce bacterial and viral contaminants in foods with emphasis on molluscan shellfish.
1. Scallan, E., R.M. Hoekstra, F.J. Angulo, R. V. Tauxe, M.A. Widdowson, S.L. Roy, J.L. Jones and P.M. Griffin. 2011. Foodborne illness acquired in the United States — major pathogens. Emerg Infec Dis 17:7–15.
2. Richards, G.P. 2001. Enteric virus contamination of foods through industrial practices: A primer on intervention strategies. J Indust Microbiol Biotechnol 27:117–125.
3. Kohn, M.A., T.A. Farley, T. Ando, M. Curtis, S.A. Wilson, Q. Jin, S.S. Monroe, R.C. Baron, L.M. McFarland and R.I. Glass. 1995. An outbreak of
Norwalk virus gastroenteritis associated with eating raw oysters. Implications for maintaining safe oyster beds. JAMA 273:466–471.
4. McIntyre, L., E. Galanis, K. Mattison, O. Mykytczuk, E. Buenaventura, J. Wong, N. Prystajecky, M. Ritson, J. Stone, D. Moreau and A. Youssef. 2012. Multiple clusters of norovirus among shellfish consumers linked to symptomatic oyster harvesters. J Food Prot 75:1715–1720.
5. DiGirolamo, R., J. Liston and J. Matches. 1970. Survival of virus in chilled, frozen, and processed oysters. Appl Microbiol 20:58–63.
6. Richards, G.P., C. McLeod and F.S. Le Guyader. 2010. Processing strategies to inactivate enteric viruses in shellfish. Food Environ Virol 2:183–193.
7. Provost, K., B.A. Dancho, G. Ozbay, R.S. Anderson, G.P. Richards and D.H. Kingsley. 2011. Hemocytes are sites of enteric virus persistence in oysters. Appl Environ Microbiol 77:8360–8369.
8. Kingsley, D.H., D.G. Hoover, E. Papafragkou and G.P. Richards. 2002. Inactivation of hepatitis A virus and a calicivirus by high hydrostatic pressure. J. Food Prot 65:1605–1609.
9. Kingsley, D.H., D.R. Holliman, K.R. Calci, H. Chen and G.J. Flick. 2007. Inactivation of a norovirus by high pressure processing. Appl Environ Microbiol 73:581–585.
10. Leon, J.S., D.H. Kingsley, J.S. Montes, G.P. Richards, G.M. Lyon, G.M. Abdulhafid, S.R. Seitz, M.L. Fernandez, P.F. Teunis, G.J. Flick and C.L. Moe. 2011. Randomized, double-blinded clinical trial for human norovirus inactivation in oysters by high hydrostatic pressure processing. Appl Environ Microbiol 77:5476–5482.
11. Lees, D. and CEN-WG6-TAG4. 2010. International standardisation of a method for detection of human pathogenic viruses in molluscan shellfish. Food Environ Virol 2:146–155.
12. Dancho, B.A., H. Chen and D.H. Kingsley. 2012. Discrimination between infectious and non-infectious human norovirus using porcine gastric mucin. Int J Food Microbiol 155:222–226.
13. Tian, P., A. Engelbrektson and R. Mandrell. 2008. Two-log increase in sensitivity for detection of norovirus in complex samples by concentration with porcine gastric mucin conjugated to magnetic beads. Appl Environ Microbiol 74:4271–4276.
14. Richards, G.P. 2012. Critical review of norovirus surrogates in food safety research: rationale for considering volunteer studies. Food Environ Virol 4:6–13.
15. Di Martino, B., C. Ceci, F. Di Profio and F. Marsilio. 2010. In vitro inactivation of feline calicivirus (FCV) by chemical disinfectants: Resistance variation among field strains. Arch Virol 155:2047–2051.
16. Lee, K.M. and H.H. Gillespie. 1973. Thermal and pH stability of feline calicivirus. Infect Immun 7:678–679.
17. Shimasaki, N., T. Kiyohara, A. Totsuka, K. Nojima, Y. Okada, K. Yamaguchi, J. Kajioka, T. Wakita and T. Yoneyama. 2009. Inactivation of hepatitis A virus by heat and high hydrostatic pressure: variation among laboratory strains. Vox Sanguinis 96:14–19.