Inactivation of Norwalk-like Viruses in Shellfish by the use of High Pressure Processing

Specific objectives are: <ol><li>To determine the time/dose parameters needed to cause oysters to take up sufficient numbers of caliciviruses (SMSLV) to be used for further experiments. <li>To determine the retention time/inactivation rate of virus within the oysters. <li>To subject oysters containing SMSLV to various time/pressure conditions to determine inactivation rates. <li>To disseminate the results of the study to the shellfish industry and to the scientific community by bulletins, brochures, meetings/conferences, and peer-reviewed scientific papers. </ol>
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Cell culture and virus stocks: The viruses used in these experiments are caliciviruses isolated from feces of sea lions on San Miguel Island, CA. The agents are not considered to be human pathogens. Viruses will be propagated in porcine kidney cell line (PK-15) at 37 deg C. Virus in quantities to be used for the exposure experiments will be grown in 150 cm2 culture vessels sufficient to provide the needed quantity of virus. This would require approximately 0-20 flasks per experiment. Oysters/husbandry: Petite Japanese oysters, Crassostrea gigas, will be held at HMSC for all experiments. The facility is supplied with flowing seawater, and is treated by sand filtration, followed by ultraviolet irradiation. Total bacterial counts typically show fewer than 20 cfu/mL of treated seawater, and previous work has shown that there have been no detectable viral agents which could replicate on the PK-15 cell line (Reno and Smith, unpublished observations). To prevent contamination of the environment from these experiments, all effluents from experimental units at HMSC are treated by filtration to 1 urn, irradiated with ultraviolet light, and treated with 2-4 mg/L C12 for 1 hour prior to release. Exposure of oysters to virus. Oysters will be exposed to virus by immersion. The virus will be propagated as described above and 105 pfu will be added per ml of water. Approximately 30-40 petite oysters will be selected for each experiment. The oysters will be held for 3 h under static conditions, and the water flow will be restarted. A sample of 6 oysters will be pooled and tested for their virus content. For processing, the oysters will be surface disinfected, shucked aseptically, and homogenized in 5 volumes of PBS. The slurry will be diluted to 1:100 with MEM-lO +50 u/mL of gentamycin, filtered through a 0.45 urn filter and titrated by plaque assay; control oysters will be exposed to cell culture medium only and tested for replicating agents. High hydrostatic pressure process: Oysters harboring virus will be transported on ice at a temperature of less than 10 deg C. The samples will be subjected to a series of pressures from 120 to 350 MPa for up to 10 mm in an isostatic press unit (Model I P-2-22-60, Autoclave Engineers Inc.). Three replicate experiments will be performed for each time/pressure combination. Oysters will be vacuum packed, and the bag will be placed into the pressure chamber. The HPP operating parameters having the least effect on oysters' texture and appearance have been done in this laboratory: 325 MPa /5mm was the maximum which would retain the flavor and texture characteristics of the oyster meats and these pressure/time combinations will be our maximum test parameters. When optimal parameters for virus inactivation have been established, runs will be made on oyster meats in commercial product containers to confirm results. Shucked meats will be placed in commercial 8 oz plastic containers and will be HPP treated. The containers will be filled with shucked oysters and water, sealed with a cap and pressure treated. Preliminary tests have shown that the standard plastic container will withstand high pressure treatment.
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The American shellfish industry contributes nearly $150 million to our economy. Unfortunately, both wild and cultured shellfish harbor human viral and bacterial pathogens due to their residence in coastal areas. Since shellfish are often consumed raw, consuming contaminated shellfish can result in severe gastrointestinal disease. Even a minor outbreak can have a ripple effect, reducing consumption not only in areas nearest the outbreak, but also regionally and even nationally. The Norwalk virus is a prime cause of shellfish food poisoning. Several methods have been used to reduce disease agents in shellfish, but each has drawbacks. Heating is effective in reducing pathogens, but can affect the taste of oysters, and the stigma of irradiation precludes its use. High pressure processing (HPP) is a heatless method which uses compression of water to inactivate microorganisms. The pressure is achieved instantaneously throughout the food and causes no significant changes to the food during processing. Studies that have been conducted on shellfish indicate the high effectiveness of HPP in inactivating bacteria and other viruses. We will utilize HPP, which has proven effective in eliminating pathogenic bacteria and viruses in other foods. Since the primary virus involved in shellfish food poisoning (the Norwalk virus) cannot be grown outside the body, we will use a similar virus of marine origin (San Miguel sea lion virus) as a surrogate for the Norwalk agent in these studies.
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We have only recently initiated studies on the survival of ocean-origin caliciviruses, namely San Miguel Sea Lion Virus (SMSV), under conditions of high hydrostatic pressure. We have performed some preliminary experiments and are beginning to gear up for a more extended series of experiments with the virus. Using virus in culture medium alone, it was found that at pressures of 310 mPa for 2 minutes, greater than 106 TCID5O/mL of virus was inactivated. Pacific oysters were also inoculated with virus by immersion and a similar experiment was carried out. It was found that an equivalent loss of virus activity was achieved at the same pressure. No virus was detected in negative control oysters which had not been exposed to exogenous virus.
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If the use of high hydrostatic pressure is capable of inactivating Norwalk-like agents such as SMSV, this will significantly help the oyster industry. The results of our previous experiments, as well as others, have found that these pressures shuck the oyster as well as inactivate large numbers of pathogenic bacteria, especially Vibrio parahaemolyticus. The inactivation of a serious human pathogen such as Norwalk agent would enhance its use in both food safety and in increasing the efficacy and thereby lowering the cost of processing oysters and possibly other shellfish.