Thermal inactivation of human norovirus surrogates in oyster homogenate
Introduction
In the United States, human norovirus (HNV) is the most common cause of acute gastroenteritis, leading to 19–21 million illnesses, 56,000–71,000 hospitalizations and 570–800 deaths each year (Hall et al., 2013). It is considered the most frequent causative agent causing more than half of foodborne diseases in the US cases each year (Scallan et al., 2011). Norovirus causes acute gastrointestinal infection with common symptoms include diarrhea, vomiting, nausea, abdominal cramping, chills, headache, dehydration and a high-grade fever (Li et al., 2013). It is highly contagious because only a few particles have the potential to cause infection (Donaldson et al., 2008). It can be spread by contaminated food or water, through person to person contact and via cross contamination surfaces (Hall et al., 2013). Raw and undercooked oysters are commonly involved in outbreaks caused by HNV; they are filter feeders and concentrate and retain viruses derived from the environment (Lees, 2000). From 1998 to 2015, 5362 foodborne outbreaks in the US were caused by norovirus, in 86 outbreaks of which oyster is the vehicle (Foodborne Outbreak Online Database (FOOD Tool), 2016). In 2016, approximately 75 people in the outer Cape Cod area in Massachusetts developed norovirus-like illness after eating raw shellfish (NoroCore, 2016). In 2002, over 100 people in Italy became ill after consuming norovirus contaminated oyster (Le Guyader et al., 2006).
It has been demonstrated that HNV can interact specifically with carbohydrate structures in the bivalve digestive organ (Le Guyader et al., 2000). Viruses cannot multiply in food or in the environment, but they can persist for several days or weeks without loss of infectivity (Seitz et al., 2011). Typical methods used to prevent bacterial growth in food products may not be effective against viruses (Jaykus, 2000). Therefore, the potential presence of HNV in oysters poses a serious health threat to consumers and is an important concern for health authorities (Hewitt and Greening, 2004). It is then essential to understand whether current seafood handling and processing can mitigate HNV survival and persistence in oysters.
Recently, Dr. Mary Estes and her research team have successfully grown human norovirus in enterocytes in stem cell–derived, nontransformed human intestinal enteroid monolayer cultures with bile (Ettayebi et al., 2016). However, before the research, the main difficulty that hampers research of HNV is there is no in vitro cell culture system or small animal model. As a result, most of research relies on viral surrogates, including feline calicivirus (FCV) and murine norovirus (MNV-1). FCV is structurally different from HNV and it is a respiratory virus and very sensitive to low pH (2.0 to 4.0) (Cannon et al., 2006; Li et al., 2012). MNV-1 has been shown to be more similar to HNV immunologically, biochemically, genetically, and molecularly. And it belongs to genus norovirus and is also resistant to acid and heat, and highly stable and persistent in the environment (Cannon et al., 2006; Li et al., 2012). However, clinical symptoms of gastroenteritis caused by MNV-1, which present as hepatitis, pneumonia, and inflammation of nervous systems, are quite different from that caused by HNV (Karst et al., 2003). More importantly, MNV-1 uses sialic acid as a functional receptor whereas HNV uses HBGA as receptors (Wobus et al., 2006; Tan and Jiang, 2010). Previous research on thermal inactivation showed that FCV and MNV-1 behaved similarly when heated at 63 °C (Cannon et al., 2006). It was reported that Tulane virus (TV), a calicivirus isolated from stools of rhesus macaques, represents a new genus, Recovirus (Farkas et al., 2008). TV can be cultivated in rhesus monkey kidney cells (LLC-MK2) and is close to HNV based on its genomic sequence (Farkas et al., 2010). More importantly, like HNV, it recognizes the type A and B HBGAs (Farkas et al., 2010). Therefore, it has the potential for use as a surrogate of HNV. In terms of thermal resistance, TV in culture medium is more heat sensitive than MNV-1 at 50 to 60 °C (Hirneisen and Kniel, 2013). However, there is no report of the stability of TV when heated in seafood matrix such as oysters.
Thermal processing is one of the most effective methods to reduce viruses in any food product. Cooking oysters thoroughly will impact organoleptic characteristics and can toughen oyster meat, which make them undesirable for consumers. Light cooking may be acceptable to some consumers, but might be insufficient to kill all enteric viruses, since most of the viruses are inside the shellfish and would not be subjected to sufficient heat for their total inactivation (Richards et al., 2010). It has been suggested that an internal temperature of 90 °C for at least 90 s is a virucidal treatment. FDA suggests that seafood is cooked to an internal temperature of 63 °C (145 °F) for 15 s (FDA, 2009), which ensures that food-borne bacteria is destroyed. Consumers, without thermometers, can rely on shells to open to determine the doneness of shellfish. However, this practice may be insufficient to reach the virucidal treatment. Previous studies on steaming mussel showed that the mean internal temperature was 83 °C when all 50 mussels were tested (Hewitt and Greening, 2006). It is also recommended by many guidelines that shucked oyster is simmered or boiled for at least 3 min (Villalba et al., 2008; Hicks, 2010). Since there is no specific regulation covering the minimum time–temperature combination for inactivating virus in contaminated oysters, establishment of proper thermal processes for inactivating HNV in a high risk food such as oysters would be essential for protecting public health.
The specific objectives of this study were to (1) determine thermal inactivation behavior of murine norovirus (MNV-1) and Tulane virus (TV) in oyster homogenate (2) test 3 min of boiling water heating efficacy on inactivation of both viral surrogates in oyster homogenate and (3) compare first-order and Weibull models to understand the kinetics of thermal inactivation behavior of two viral surrogates.
Section snippets
Viruses and cell lines
MNV-1 and TV were propagated in RAW 264.7 and LLC-MK2 respectively. Raw 264.7 cells were cultured in high-glucose Dulbecco's modified with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies) at 37 °C under a 5% CO2 atmosphere. MK2-LLC cells were cultured in M199 medium (Mediatech, Manassas, VA) with 10% heat-inactivated FBS (Life Technologies) and penicillin G (100 U/ml) and streptomycin (100 μg/ml) at 37 °C under a 5% CO2 atmosphere. To prepare MNV-1 stock, confluent RAW 264.1
Internal temperature of oyster homogenate and inactivation effects by boiling water
The mean temperature of oyster homogenate prior to treatment was 18 °C. After 1, 2, and 3 min of heating, the internal temperature reached 71, 82, and 91 °C respectively. After boiling for 262 s, the mean internal temperature was 95 °C and maintained at that level (Fig. 1). Compared with previous thermal profile described by Hewitt and Greening (2006), at 1, 2, and 3 min, the mean internal temperature reached 60, 80, and 90 °C respectively when heating mussels in boiling water, which is similar
Conclusion
Our results suggest that boiling oyster homogenate for 3 min may be effective to inactivate HNV to below 1 log10 PFU/ml based on the results of two surrogates, MNV-1 and TV. Among the three temperature tested (58 and 63, 67 °C), inactivation at a higher temperature has a faster inactivation rate compared to lower temperatures. The results also indicate MNV-1 was more heat resistant than TV in oyster homogenate, especially >58 °C. Lastly, the result of R2, RMSE and MSE demonstrated that the
Acknowledgement
This project was supported by Delaware Sea Grant, SFA-1 (NA14OAR4170087 – DESG 2014-2016) and partially by the National Institute of Food and Agriculture, U.S. Department of Agriculture (USDA) (grant 2011-68003-30005).
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