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Inactivation of Enteric Foodborne Viruses in High Risk Foods by Non-Thermal Processing Technologies

Chen, Haiqiang
University of Delaware
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This study will focus its efforts on the most significant foodborne enteric viruses, human norovirus, although hepatitis A virus and rotavirus will also be studied. The overall goal is to identify effective non-thermal processing technologies and to optimize processing parameters to destroy these viruses in high risk foods, shellfish (oysters and clams), fresh and frozen berries (strawberries, raspberries and blueberries), berry purees, green onions, and salsa. In addition, the efficacy of these non-thermal processing technologies will be tested on pathogenic bacteria that cause large outbreaks in these high risk foods (Vibrio parahaemolyticus in shellfish and Escherichia coli O157:H7 and Salmonella in other products). The effect of the processing technologies on the quality of high risk foods will be evaluated. The knowledge gained will be disseminated through education curricula and outreach programs.
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NON-TECHNICAL SUMMARY: Viruses account for more than 67% foodborne illnesses worldwide. Commonly implicated foods include raw or under-cooked shellfish, produce and produce products such as berries, green onions, and salsa. None of the decontamination methods investigated thus far have been shown to effectively control foodborne viruses in high risk foods. Therefore, there is an urgent need to develop novel processing technologies to inactivate viruses. The overall goal of this project is to identify effective processing technologies and to optimize processing parameters to destroy foodborne viruses in high risk foods and disseminate the knowledge through education and outreach.

Through this project, we expect to 1) develop effective processing technologies to control foodborne viruses in high risk foods; 2) understand the mechanism of viral inactivation by these processing technologies; 3) develop two courses and disseminate course contents to institutions across the United States; and 4) provide outreach education and training to industry and educators regarding foodborne viruses and food safety impacts of these processing technologies on high risk foods. Through integration of research, education and extension, our study will enhance the safety of high risk foods and reduce foodborne viral illnesses

APPROACH: We will first determine whether an in vitro cultivable enteric calicivirus, the Tulane virus, can be used as a surrogate for human norovirus and develop a culture system to assess the survival of human norovirus. Four non-thermal technologies: 1) high pressure, 2) irradiation, 3) UV, and 4) washing with sanitizers, will be studied on their inactivation of the viruses in high risk foods. Effective technologies for specific foods will be identified and processing parameters for virus inactivation will be established. Mathematical models will be used to predict the inactivation of the viruses. The mechanism of viral inactivation by these four processing technologies will be studied. We will investigate whether viral capsid proteins, viral genome and receptor binding activity are damaged or denatured by the processing technologies. We will also examine the damage of the virion by electron microscope. In addition, the efficacy of these technologies will be tested on the pathogenic bacteria that are related to these high risk foods. The effect of these processing technologies on quality attributes of the high risk foods will be evaluated. Sensory quality, important nutrients in the high risk foods, color, texture, and other important properties will be evaluated. We will develop two courses ("Non-Thermal Food Processing Technologies" and "Foodborne Viruses and Food Safety") and train students in non-thermal processing technologies, food virology, and food safety. Outreach education and training to industry and educators regarding foodborne viruses and food safety impacts of non-thermal technologies on high risk foods will be provided. A variety of approaches to develop and implement the best outreach strategies will include 1) assessment of industry and educators attitudes and knowledge about non-thermal processing and its applications; 2) development of training courses, e.g. webinars and on-site presentations; 3) development of appropriate brochures, pamphlets, or other outreach alternatives as directed by the assessment and 4) evaluation of outreach strategies. The educational resources developed for the target audiences during the program will reflect the results of the research and assessment efforts of the project. Where possible, evaluations will follow Kirkpatrick's four level models for evaluating training.

PROGRESS: 2012/02 TO 2013/01
OUTPUTS: Tulane virus (TV) as a surrogate for human noroviruses (HuNoV): TV was sensitive to heat (63C for 5 min or at 56C for 30 min), UV (60 mJ/cm2) and chlorine (300 ppm). It was stable at pH 3-8 and resistant to ≤ 40% of ethanol, but could be inactivated by 50-70% ethanol. Viral inactivation mechanism: a) High hydrostatic pressure (HHP)- VLPs were more resistant to HHP in their ability to bind type A than type B and O HBGAs. The 23-nm VLPs appeared to be much more stable than the 38-nm VLPs; b) E-beam irradiation- E-beam affected the structure of murine norovirus (MNV-1) and vesicular stomatitis virus. It degraded viral genomic RNA and viral proteins. Inactivation of viruses: a) Pressure inactivation of HuNoV- Pig gastric mucin-conjugated magnetic beads (PGM-MB) were used to bind and collect infectious HuNoV followed by qRT-PCR to quantify it. Using this approach, GI.1 HuNoV was found to be more resistant to HHP than the GII.4 strain. Treatment at 600 MPa achieved a > 4.1 log reduction of GI.1 NoV, which agrees with the results from a recent human volunteer study. HHP inactivation of the two HuNoV strains increased as sample temperature decreased from 35 to 1C. Both strains were more resistant to pressure under acidic conditions; b) E-beam irradiation of MNV-1- Less than 1 log virus reduction was achieved at 2, 4, and 6 kGy for buffer, cabbage, and strawberries, respectively. Inactivation of pathogenic bacteria: a) HHP inactivation of vibrios in oysters- Cold storage at -18, 4 and 10C, prior to HHP, decreased V. parahaemolyticus or V. vulnificus by 1.5 - 3.0 log MPN/g, but did not increase their sensitivity to subsequent HHP treatments. V. parahaemolyticus populations in HHP-treated oysters gradually decreased during post-HHP ice or frozen storage; b) Pulsed light (PL) inactivation of E. coli O157:H7 on blueberries- Inoculated berries were treated by PL alone (dry PL) or immersed in agitated water during the PL treatment (wet PL). The wet PL treatment reduced E. coli O157:H7 on blueberries by > 5 log CFU/g. Both dry and wet PL treatments were more effective than washing with 10 ppm chlorinated water; c) PL inactivation of E. coli O157:H7 on green onions- For dip-inoculated green onions, 60s wet PL treatment was comparable with 100 ppm chlorine washing. PL combined with surfactant (SDS) was more effective than PL alone; d) Sanitizer wash for blueberries- Wash time and storage temperature influenced the reduction of Salmonella Typhimurium. Survey: The survey to assess the industry knowledge and attitudes regarding non-thermal processing has been completed. The database included 113 participants. Both shellfish and produce respondents had very low knowledge scores, 51% and 48%, respectively; indicating the need for education and outreach efforts. While 100% of the produce processors had established food safety training programs, only 50% of the shellfish processors identified themselves as having one. The majority were interested in receiving training and information on non-thermal processing. The survey to assess food safety educator knowledge and attitudes regarding non-thermal processing was recently completed.
PARTICIPANTS: 1. PI: Haiqiang Chen (University of Delaware) 2. Co-PIs: Brendan Niemira (USDA, ARS, Eastern Regional Research Center), Changqing Wu (University of Delaware), Gulnihal Ozbay (Delaware State University), Xi Jason Jiang (Cincinnati Children's Hospital Medical Center), Jianrong Li (The Ohio State University), Joshua Gurtler (USDA, ARS, Eastern Regional Research Center), Ken Lee (The Ohio State University), Lori Pivarnik (University of Rhode Island), Randy Worobo (Cornell University), and Yi-Cheng Su (Oregon State University) 3. Research Support Specialist: John J. Churey (Cornell University) 4. Post-doctoral fellows: Xinhui Li (University of Delaware), Christina Quigley (Cincinnati Children's Hospital Medical Center), and Alison Lacombe (USDA ARS) 5. Graduate Students: a) University of Delaware: Mu Ye, Yaoxin Huang, Robert Sido, Chuhan Liu, Wenqing Xu, and Yingying Li; b) Oregon State University: Sureerat Phuvasate; c) The Ohio State University: Erin Dicaprio, Fangfei Lou and Yue Duan; d) Cornell University: Carmen Wickware, Giselle Guron, and Claire Zoellner 6. Undergraduate Student: Robert Sido (summer student, University of Delaware), Melissa Ehrich (University of Delaware), Marissa Chou (summer student, Cincinnati Children's Hospital Medical Center)
TARGET AUDIENCES: Two courses have been developed and are currently taught by co-PI, Dr. Jianrong Li. Course 1 - Food Safety and Public Health: Food safety is an important component of public health. Course 2 - Advanced Food Microbiology II - Food Virology and Immunology.

IMPACT: 2012/02 TO 2013/01
TV as a surrogate for HuNoV: Although TV behaved similarly to various HHP conditions as MNV-1and the two stains of HuNoV (GI.1 and GII.4), it might not be an optimal surrogate for HuNoV, as it is slightly more sensitive to pressure than the two HuNoV strains. The likely order of pressure resistance is: GI.1>MNV-1>GII.4>TV. Therefore, MNV-1 may be considered the better surrogate for GII.4. Viral inactivation mechanism: a) HuNoV VLPs- Using VLPs as a model to understand the stability of HuNoV, we demonstrated that the HuNoV capsid is highly resistant to HPP; b) E-beam- The mechanism of E-beam inactivation of viruses is similar to that of gamma irradiation. Inactivation of viruses: a) HHP- HHP could be applied as a potential intervention for inactivating HuNoV in raw oysters. To effectively inactivate HuNoV GI.1, the pressure may need to exceed > 500 MPa, which is much higher that pressure levels currently used by the oyster industry (NoV, wMPa). The impact of such high pressures on sensorial properties of oysters remains to be determined. Low treatment temperature could be used to reduce the pressure level needed for effective inactivation of HuNoV; b) Binding assay- Binding to PGM-MB, followed by RT-PCR detection, can be explored as a practical means of evaluating the infectious and non-infectious states of NoV. The assumption is that non-infectious HuNoV was not able to bind to PGM; c) E-beam- In foods, MNV-1 is difficult to inactivate by E-beam irradiation due to the protective effect of the food matrix and small size and the highly-stable viral capsid. The application of E-beam to inactivate HuNoV in foods is probably unlikely. Inactivation of pathogenic bacteria: a) HHP inactivation of vibrios in oysters- Effective combinations of pressure treatment and cold storage were identified. HPP at (1) 300 MPa for 2 min at 21C, followed by 5-day ice storage or 7-day frozen storage, or (2) HPP at 250 MPa for 2 min at 21C, followed by 10-day ice or 7-day frozen storage, completely inactivated V. parahaemolyticus in whole-shell oysters (> 7 log reductions). The combination of HHP at a relatively low pressure (e.g., 250 MPa), followed by short-term frozen storage (7 days), could potentially be applied by the shellfish industry as a post-harvest process to eliminate V. parahaemolyticus in oysters; b) PL treatment- The wet PL treatment could be a promising alternative to traditional chlorine washing for blueberries intended for frozen storage and green onions, to avoid the use of chemicals. Since berries destined for the fresh market are usually not washed, the dry PL treatment can potentially be used to reduce the level of pathogens on fresh berries. In addition, SDS could be used to enhance PL inactivation of E. coli O157:H7 on green onions; c) Sanitizer wash for blueberries- Effective washing solutions and treatment times were identified. The combination of 200 ppm H2O2 and 0.5% SDS had greater efficacy in inactivating S. Typhimurium after a 5 min wash (4.0 log CFU/g reduction) than a 1 min wash (3.2 log CFU/g reduction). Treated blueberries stored at lower temperatures significantly enhanced visual appearance and decreased mold growth.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Education and Training
Viruses and Prions