Progress 09/15/05 to 12/14/09
OUTPUTS: Four randomized, double-blinded, placebo-controlled clinical trials were conducted to assess the potential of high hydrostatic pressure processing (HPP) to inactivate Norwalk virus (NV: G1.1), both in water and in oysters. Because there are no suitable in vitro methods or practical laboratory animals for assaying inactivation of human norovirus strains, this work required human feeding studies. Volunteer recruitment was conducted through self-selection (flyer advertisements posted throughout Emory Hospital and campus) and referrals from other study subjects. Enrollment in the study occurred from September 2007 to October 2009. Norwalk virus inoculum (8FIIb) prepared from stool filtrates made from a previously NV-infected volunteer was used to inoculate water and oysters. Prior to contamination, oysters were screened for total and fecal coliform bacteria, and the presence of NV, and hepatitis A virus. An initial 400 MPa for 5 min was performed to ensure inactivation of any vibrio bacteria present, and provide an intact oyster for injection of Norwalk virus 8FIIb. 1/3 ml NV inoculum was injected into each of 3 oysters. Water and oysters (Crassostrea virginica) were injected with a combined dose of 1.0 x 104 genomic copies of the Norwalk virus 8FIIb strain. Study phases were based on HPP pressure and temperature parameters and included the following: Phase 0: 400 MPa for 5 minutes at 25 C for water seeded with NV, phase 1: 400 MPa for 5 minutes at 25 C for NV-seeded oysters, phase 2: 600 MPa for 5 minutes at 5 C for NV-seeded oysters, and phase 3: 400 MPa at 5 C for NV-seeded oysters. These treatments were applied using a Quintus 35-L food press (QFP 35L-600; Flow International Corporation, Avure Technologies Inc., Kent, WA). For all studies, two minutes prior to water or oyster consumption, volunteers ingested 2 oz. of sodium bicarbonate dissolved in water to reduce stomach acidity. Volunteers within the water pilot study received either the placebo (untreated water seeded with 10,000 genomic copies of 8FIIb NV) or HPP treated water with 10,000 genomic copies of 8FIIb NV. Volunteers within phases 1-3 received either the placebo (3 untreated oysters with a combined NV (8FIIb) dose of 10,000 genomic copies) or 3 HPP-treated oysters with a combined NV (8FIIb) dose of 10,000 genomic copies. Oysters were consumed with the option of eating saltine crackers and/or reduced fat chocolate milk, to facilitate the complete consumption of the oysters/oyster juice without affecting the stomach gastric environment. For all phases, treatment and placebos were supplied in identical sterile vials. A subsequent administration of dissolved sodium (2 oz) bicarbonate was given 5 minutes following dosing to further reduce stomach acidity. The results of the work were presented at the Interstate Seafood Seminar held at Virginia Beach, Virginia, at the International Shellfish Conference, held at Prince Edward Island, Canada, and at meetings of the American Society for Virology, Vancouver, Canada and Bozeman, Montana. The audiences included employees from the Food and Drug Administration (DDA), state health regulatory officials, physicians and university faculty. PARTICIPANTS: Dr. George Flick served as principle investigator on this project. Ms. Shelia Holliman performed selected microbiological analyses on project samples. Ms. Angela Correa was responsible for the outreach component of the project including news releases and technical reports. Drs. Juan Leon and Christine Moe, Emory University School of Public Health supervised all clinical trials. The clinical research team was composed of Dr. G. Marshall Lyon and research nurse Gwen Abdulhafid, who worked with the volunteers. Drs. David Kingsley and Gary Richards, USDA, ARS performed quality control testing of the shellfish, contaminated shellfish with noroviruses, pressure treated the shellfish, and performed virus analyses. Ms. Gloria Meade and Mr. Michael Watson, USDA, ARS assisted in bacterial and viral analyses. There were multiple opportunities for professional development that were provided by the project: 1) investigators presented their work at national meetings; 2) undergraduate and graduate students were trained in clinical trial methodology and laboratory analyses; 3) undergraduate and graduate students had the opportunity to present their work in local venues. TARGET AUDIENCES: The target audience for this work is food companies that process molluscan seafood that might contain norovirus. It is also of interest to consumers of food products where norovirus contamination could result in foodborne illness, especially individuals who consume raw molluscan seafood. PROJECT MODIFICATIONS: Not relevant to this project.
In all 3 phases of the clinical trials involving oysters, some of the volunteers receiving control oysters (injected with Norwalk virus and not high pressure processed) did become infected with Norwalk virus, indicating that the oysters were infectious. Some of the human subjects in Phase I and III of the trials that received Norwalk-injected oysters that were subsequently high pressure processed at 400 MPa for 5 minutes at temperatures of 25 and 5 C (trials I and III respectively) also became infected with Norwalk virus. Hence, this research demonstrated that 400 MPa for 5 minutes is not sufficient (even at a range of temperatures) to inactivate Norwalk virus within oysters. However, of the ten human subjects ingesting oysters injected with Norwalk virus that were subsequently processed with 600 MPa of high pressure for five minutes at temperatures ranging from 4 to 7 C, none became infected with Norwalk virus. This result indicates that this combination of pressure/time/temperature is effective in inactivating the virus within an oyster. Six hundred MPa pressure is greater than what is currently being commercially applied by the shellfish industry; thus the commercial value of this treatment process for the raw oyster market remains to be determined. The results of this research could be used by seafood industries that further process oysters for markets other than the raw oyster market. These processors tend to inventory large amounts of product for subsequent use during the year. Typically, if a virus contamination occurs in an approved oyster growing area, any oysters in inventory from that area must be destroyed since customary cooking procedures (such as grilling, deep frying and baking) do not destroy the Norwalk virus. High pressure processing is one technology that can inactivate the Norwalk virus without destroying or substantially altering the physical properties of oysters. This research indicates that murine norovirus (MNV-1) is more susceptible to deactivation by high pressure processing than is Norwalk virus (8F11b); hence, MNV-1 is not an ideal surrogate virus for human Norwalk virus in pressure studies. The outreach objectives of this project were satisfied in part through the following oral presentations made by Dr. David Kingsley: 1)Norovirus:Human Health and Food-borne Implications. Anne Arundel County Health Laboratory. Annapolis, MD. Oct 2009; 2)Detection, Isolation, and Persistence of Viruses within Bivalve Mollusks. University of Bari, Bari, Italy. May 2009; 3) Quest for a Food-borne Virus Intervention Strategy. ARS-Purdue. US Sino Food Safety Research Center Meeting. Blue Bell, PA. Nov 2008; and 4)High Pressure Inactivation of Food-Borne Viruses. High Pressure Bioscience & Biotechnology Conf. Scripps Oceanographic Istitute. La Jolla, CA. Sept 2008.
- Kingsley,D. H. J. S. Leon, G. P. Richards. G. Flick, G. M. Lyon, G. Abdulhafid, J. Sobolik, A. Creadore, S. Seitz, E. Smith, M. Fernandez, and C. Moe. (2010). High Pressure Processing Inactivates Human Norovirus within Oysters. 29th Annual Meeting of American Society for Virology, Bozeman MT pp.21-22.
Progress 09/15/08 to 09/14/09
OUTPUTS: This study was conducted in three phases. In each phase, oysters were exposed to sufficiently high pressures (400 MPa for five minutes) to cause the abductor muscle to release. This allowed the oyster to be injected with Norwalk virus strain 8FIIb. After injecting, some of the oysters were exposed to a second high pressure processing treatment. The specific pressure/time/temperature combination applied varied for each phase of the research. After the high pressure processing treatment was applied to the Norwalk-injected oysters they were shipped to Emory University, where feeding trials were conducted on human subjects to determine if the virus was successfully inactivated. Detailed screening was conducted to identify human subjects eligible for the test protocol (subjects had to show genetic susceptibility to infection by Norwalk virus, be healthy, and between the ages of 18 and 55). Some subjects received oysters that were injected with Norwalk virus and not exposed to high pressure processing (control group) while some subjects received injected oysters that were subsequently high pressure processed. In Phase I of this project, Norwalk virus-injected oysters were subjected to high pressure processing at 400 MPa at room temperature for a duration of five minutes. Based on the results of the trials conducted with human subjects with these oysters, which indicated that this treatment was not sufficient to completely deactivate the Norwalk virus, Phase II of the study was initiated. In Phase II, oysters were again injected with Norwalk virus and then exposed to 600 MPa of pressure at 4 to 7 C for a duration of five minutes. Based on the human subjects trials, this treatment did completely deactivate the virus. After Phase II, a third phase of research was initiated in which oysters injected with Norwalk virus were exposed to high pressure processing at the lower pressure level used in Phase I(400 MPa), and at the lower temperature (4 to 7 C) used in Phase II. The duration of the pressure application for Phase III was the same as in Phases I and II (five minutes). Again, trials were conducted at Emory University feeding both high pressure processed (treated) and non high pressure processed (untreated) Norwalk-injected oysters to human subjects. The results of the work conducted to date were presented at the Interstate Seafood Seminar held at Virginia Beach, Virginia, at the International Shellfish Conference, held at Prince Edward Island, Canada, and at the meeting of the American Society for Virology, Vancouver, Canada. The audiences included employees from the Food and Drug Administration (FDA), state health regulatory officials, physicians and university faculty. PARTICIPANTS: Dr. George Flick served as principle investigator on this project. Ms. Shelia Holliman preformed selected microbiological analyses on project samples. Ms. Angela Correa was responsible for the outreach component of the project including news releases and technical reports. Drs. Juan Leon and Christine Moe, Emory University School of Public Health supervised all clinical trials. The clinical research team was composed of Dr. G. Marshall Lyon and research nurse Gwen Abdulhafid who worked with the volunteers. Drs. David Kingsley and Gary Richards, USDA, ARS performed quality control testing of the shellfish, contaminated shellfish with noroviruses, pressure treated the shellfish, and performed virus analyses. Ms. Gloria Meade and Mr. Michael Watson, USDA, ARS assisted in bacterial and viral analyses. There were multiple opportunities for professional development that were provided by the project: 1) investigators presented their work at national meetings; 2) undergraduate and graduate students were trained in clinical trials methodology and laboratory analysis; 3) undergraduate and graduate students had the opportunity to present their work in local venues. TARGET AUDIENCES: The target audience for this work is food companies that process molluscan seafood that might contain Norwalk virus. It is also of interest to consumers of food products where Norwalk virus contamination could result in foodborne illness, especially individuals who consume raw molluscan seafood. PROJECT MODIFICATIONS: Not relevant to this project.
In all three phases of the research, some of the human subjects receiving control oysters (injected with Norwalk virus but not high pressure processed) did become infected with Norwalk virus. This result demonstrated that the oysters were infectious. Some of the human subjects in Phase I and III trials that received Norwalk virus-injected oysters that were subsequently exposed to 400 MPa for five minutes at temperatures of 25 and 5 C, respectively also became infected with Norwalk virus. Hence, it was demonstrated that 400 MPa does not completely inactivate Norwalk virus within oysters. However, of the ten human subjects receiving oysters injected with Norwalk virus that were treated with high pressure at 600 MPa, none became infected with Norwalk virus, indicating that this level of pressurization was effective in completely inactivating the virus within an oyster. 600 MPa is a higher pressure than what is currently being applied in commercial applications by the shellfish industry; thus the commercial value of this treatment process for the raw oyster market remains to be determined. The results of this research could be used by seafood industries that further process oysters for markets other than the raw oyster market. These processors tend to inventory large amounts of product for subsequent use during the year. Typically, if a virus contamination occurs in an approved oyster growing area, any oysters in inventory from that area must be destroyed since customary cooking procedures (such as grilling, deep frying and baking)do not destroy the Norwalk virus. High pressure processing is one technology that can inactivate the Norwalk virus without destroying or substantially altering the physical properties of oysters. This research indicates that murine norovirus (MNV-1) is more susceptible to deactivation by high pressure processing than is Norwalk virus (8F11b); hence, MNV-1 is not an ideal surrogate virus for human Norwalk virus in pressure studies.
- Flick, GJ (2009). High pressure processing: thought and substantial research required. Fleischwirtschaft International. 24(3):14, 16-18.
- Leon JS, Moe CL (2006) Role of viruses in foodborne disease. Food consumption and disease risk: consumer-pathogen interactions (Potter M. ed.). Woodhead publishing in food science, technology and nutrition. Baltimore.
- Leon JS, Souza M, Wang Q, Smith ER, Saif LJ, Moe CL (2008) Immunology of norovirus infection. Immunity against mucosal pathogens (Vajdy M. ed.). Springer Science. Boston.
Progress 09/15/07 to 09/14/08
OUTPUTS: To address Objective (1) "identify one or more high hydrostatic pressure process schedules that result in the inactivation of noroviruses," we began the human norovirus challenge studies at Emory University. The goal of these studies was to assess whether high pressure processing (HPP) was successful at inactivating human norovirus on oysters and would result in no infection when HPP treated norovirus (NoV) seeded oysters were fed to human volunteers. We have secured all human ethical and regulatory approvals with our Institutional Review Board and set up a Data Safety Monitoring Board (DSMB) to assure the safety of our volunteers. This study was divided into two phases: 1) A pilot study and 2) the actual oyster study. In the pilot study, we enrolled 8 volunteers and confirmed that administration of our NoV dose (8FIIB Norwalk virus at 3000 genomic copies) to our volunteers resulted in NoV infection (as defined by detection of NoV in stool or vomitus samples by RT-PCR or seroconversion to NoV antigen by ELISA). We also found that HPP treatment (400 mPa, 5 minutes, 25C) of NoV seeded water samples resulted in no infection in two volunteers. After completion of the pilot study, we proceeded to the oyster study (with approval of our DSMB). In the oyster study, we enrolled 9 volunteers and fed then (in a random double blind manner) norovirus seeded oysters that had either been treated or not treated with HPP. The HPP condition for this first step was 400 mPa for 5 minutes at 25C. We found that these HPP treatment conditions were ineffective as 3 of 5 volunteers who received NoV seeded and HPP treated oysters became infected. In total, we have dosed 16 volunteers and they are all healthy. Our next steps are to assess whether other HPP conditions (e.g. lower temperatures, higher pressures) are effective at inactivating NoV on oysters. PARTICIPANTS: Dr. George Flick is principle investigator for this project. Dr. Daniel Holliman is no longer involved in this grant due to his death in April 2008. Ms. Laura Douglas is responsible for sample preparation and high pressure processing. Ms. Dianne Bourne preforms selected microbiological analyses on project samples. Dr. Juan Leon, Emory University School of Public Health is supervising clinical trials. Dr. Christine Moe Emory University School of Public Health is supervising viral analyses on product and patient samples. Ms. Angela Correa, also listed on the grant, will supervise the outreach component of the project including news releases, technical reports and journal articles. Cooperators include Dr. David Kingsley, USDA, ARS and Dr. Gary Richards, USDA, ARS. TARGET AUDIENCES: The target audience for this work is consumers of food products where Norwalk virus contamination could result in food borne illness. The project is of particular interest to individuals who consume raw molluscan seafood. The project is ongoing and it is premature at this point to release or disseminate any project results. PROJECT MODIFICATIONS: Not relevant to this project.
Our human challenge studies have shown that HPP treatment of norovirus seeded oysters at 400 mPa, 5 min, and 25C would not be effective for inactivation of norovirus in oysters. Therefore these conditions would not be useful for the oyster or shellfish industry. We are continuing to investigate other HPP conditions that the shellfish industry could use to make norovirus contaminated oysters safe for consumer consumption.
- No publications reported this period
Progress 09/15/06 to 09/14/07
OUTPUTS: Murine norovirus (MNV-1)was evaluated for susceptibility to high pressure processing. Working stocks of MNV-1 were prepared. Oysters (Crassostrea virginica) were harvested from an approved area in Mobile Bay, AL. 200 oysters were placed into a depuration flume for 2 wks then transferred to a flume with single-pass UV-treated natural seawater maintained at 21-30C. Three days before virus accumulation, 26 oysters were placed in the accumulation tank to acclimate them. MNV-1 (5.6 x 10-9 PFU for trials 1 and 2 and 2.12 x 10-8 PFU for trial 3) was added to 4 L of sterile reverse-osmosis water and continually mixed at 4C. Peristalic pumps provided an overall concentration within the accumulation tank of approximately 7,000 PFU/ml for trials 1 and 2 and 660 PFU/ml for trial 3. After 24 h, the oysters were divided into eight groups of three oysters each and shucked into sterile cups. For temperature and direct-inactivation experiments, 1.5-ml aliquots of MNV-1 in DMEM with 10% FBS
were placed in vacuum pouches and vacuum/heat sealed. Samples were then subjected to pressures from 250 MPa-450 MPa at specified starting temperatures. For MNV-1-contaminated oysters, shucked samples were transferred into pouches and heat sealed. Pressurization of oyster samples was performed using a Quintus 35-L food press. Samples were pressurized at 300, 325, 350, 375, and 400 MPa for 5 min at 5C. After processing, the refrigerated samples underwent virus extraction and assay. For MNV-1 stock virus in DMEM, samples were assayed using confluent six-well dishes. Inoculation with 0.5 ml of MNV-1 was carried out for 2 h at 37C followed by overlay with 2 ml of modified Eagle medium containing 1.5% low-melting-point agarose with 5% FBS, 2 mM Gluta-MAX-1, 100 U of penicillin, and 100 μg/ml of streptomycin sulfate. After 3 days of incubation, plaques were visualized by staining. For MNV-1 extraction from contaminated oysters and plaque assay, three shellfish per treatment group were
removed from sealed pouches, placed in 50-ml conical tubes, and centrifuged to facilitate separation of oyster meat from oyster liquor. Nonpressurized, MNV-1-contaminated (positive) controls were also tested. Virus extractions were performed by adding oyster meats (three oysters per treatment group) to phosphate buffer (up to 100 ml) and homogenizing in a laboratory blender. Homogenized extracts were centrifuged and the supernatant was neutralized with 2 N HCl. Tenfold serial dilutions were made in Earle's balanced salt solution, and plaque assays were performed in triplicate using confluent monolayers of RAW cells. For undiluted and 1:10-diluted oyster homogenates, 2 ml of extract or 2 ml of dilution was used to infect 100-mm confluent tissue culture dishes. For homogenate dilutions of 1:100 or greater, 0.5 ml was used to infect individual wells of a six-well dish. Plaques were visualized using neutral red staining 3 days postinfection.
PARTICIPANTS: Dr. David Kingsley, Research Virologist, USDA ARS, Delaware State University.
Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20C) the virus was inactivated over a pressure range of 350-450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log10 PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5C; a 5-min pressure treatment of 350 MPa at 30C inactivated 1.15 log10 PFU of virus, while the same treatment at 5C resulted in a reduction of 5.56 log10 PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0-150 s and 0-900 s at 5C and 20C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5C was sufficient to inactivate 4.05 log10 PFU. This work is the first demonstration that norovirus can be
inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.
- Kingsley, D.H., D.R. Holliman, K.R. Calci, H. Chen, and G.J. Flick. 2007. Inactivation of a norovirus by high-pressure processing. Applied and Environmental Microbiology. 73(2):581-585.
Progress 09/15/05 to 09/14/06
The first phase of this project has been completed. In collaboration with Dr. David Kingsley at Delaware State University, murine norovirus was propagated in cell culture and subjected to high hydrostatic pressure processing in the culture medium. Further collaboration with Dr. Kevin Calci of the FDA enabled high pressure processing of the virus in oysters. Culture techniques, high pressure packaging, and oyster contamination methods were initially refined until the best possible results could be obtained. In both culture medium and oysters, the virus was subjected to various pressure levels, pressure hold times, and pressure processing temperatures, and exhaustive combinations of each of these variables were also explored. Graphic inactivation curves were generated from this data across the specific variables under study. Results consistently revealed a non-linear inactivation pattern showing a "tailing" effect, though complete inactivation could also be consistently
achieved at varying pressure levels depending on the other variables involved. The virus was somewhat more resistant to pressure in oysters as opposed to culture medium, as expected. Higher pressures and longer hold times also increased inactivation as predicted. In keeping with the behavior of other caliciviruses, inactivation was improved with low temperatures. Temperatures above ambient also demonstrated increased inactivation. Depending on these variables, complete inactivation was achieved along a range of 300 to 450 MPa. Subsequent phases of the project are set to begin within the next few months. The mouse model for murine norovirus will be utilized to determine infectivity levels of pressure-treated virus within oysters. Dr. Holliman will conduct this phase. Equipment for this portion of the project (barrier isolation units) has been obtained and cell cultures propagated for virus culture. Dr Calci will perform oyster contaminations. The human feeding phase of the project,
under the direction of Dr. Christine Moe at Emory University, will also be initiated soon. For this part of the study, Dr. Kingsley will contaminate oysters with a human norovirus strain, Dr. Holliman will subject the oysters to high pressure processing, and Dr. Moe will supervise the human feedings at Emory University facilities. Inactivation parameters for a human norovirus will thus be determined.
The murine norovirus was only recently discovered and is the only known norovirus for which an animal model and cell culture exist. The validity of this virus as a surrogate for human norovirus is yet to be fully characterized. The first phases of our study will determine the high pressure inactivation character of the virus, and the usefulness of the animal model in demonstrating non-infectivity of pressure inactivated virus, as well as the necessary pressure parameters to achieve these goals. Data from this portion of the study will guide the human feeding study, and the high pressure inactivation character of both human and murine norovirus can then be compared. A specific validation of murine norovirus as a human norovirus surrogate will thus be achieved. The human feeding study results will be directly applicable to the oyster industry to guide the parameters of high pressure processing needed to decrease the incidence of oyster-borne norovirus infections and
- No publications reported this period