An Integrated Approach to Prevent and Minimize Foodborne Enteric Viruses in Vegetables and Fruits.

<p>NON-TECHNICAL SUMMARY:<br/> This special emphasis NIFSI project directly addresses the most prevalent cause of food illness with a strategy to prevent and minimize the foodborne enteric viruses in vegetables and fruits. Foodborne viruses are the dominant causative agents of fresh produce associated disease outbreaks. However, the ecology and survival of enteric viruses in fresh produce is poorly understood. None of the decontamination methods investigated so far have been shown to effectively eliminate foodborne viruses in fresh produce. No major extension and outreach effort has focused on the highly relevant challenge of virus contamination in fresh produce. In this project, we propose an integrated approach to prevent and minimize the foodborne enteric viruses in vegetables and fruits. Using cultivable murine norovirus and hepatitis A virus as the models, we will
determine the attachment, internalization, and survival of foodborne viruses in vegetables and fruits, and develop novel sanitizers and non-thermal processing technologies to remove and inactivate enteric viruses in fresh produce. Our extension effort will focus on disseminating these new knowledge and practices to stakeholders and the fresh produce industry with a goal of improving practice and implementing these new preventive strategies and interventions. Furthermore, we will incorporate these new knowledge and practices into the higher education system, develop Foodborne Viruses and Food Safety and Food Virus Education Module, and train the next generation of food safety professionals in the US. Our project will not only fill a major gap in our understanding of the interaction of virus and fresh produce, but also will result in the development and implementation of novel strategies
and interventions to prevent and minimize the foodborne viruses. In the long term, our efforts will improve the safety of vegetables and fruits through integration of research, extension, and education.
<p>APPROACH:<br/> We will use cultivable murine norovirus (NoV) and hepatitis A virus (HAV) as models to understand the attachment, internalization, and survival of foodborne viruses in vegetables and fruits, to develop novel sanitization and process technologies to inactivate the viruses, and to minimize virus contamination in fresh produce via extension, outreach and education programs. Hepatitis A virus will be propagated in fetal rhesus monkey kidney (FRhK-4) cells. Murine norovirus will grow in murine macrophage (RAW 264.7) cells. To determine the dynamics of virus attachment to fresh produce, NoV and HAV will be inoculated onto lettuce and strawberries, mixed and rinsed by distilled water with gentle agitation, and the remaining viruses in fresh produce will be quantified by plaque assay. Subsequently, we will determine the efficiency of virus transfer from hands,
contact surfaces, and packaging materials to vegetables and fruits. To investigate the viral internalization in plant tissue, we will use the following virus-food combinations: HAV-green onions and NoV-lettuce. The actively growing lettuce and green onion will be inoculated with NoV and HAV respectively. The internalized viruses will be quantified by real-time RT-PCR. The internalized viruses will be visualized using confocal or transmission electron microscopes. To develop novel sanitization methods for removing viruses from fresh produce, we will systematically screen effective sanitizer(s) by combination of various sanitizers and/or surfactants. To inactivate the internalized viruses in vegetables and fruits, we propose to use two non-thermal processing technologies (E-beam and high pressure processing). Our extension and outreach effort will focus on disseminating these new knowledge
and practices to stakeholders and the fresh produce industry with a goal of improving practice and implementing these new preventive strategies and interventions. Our education effort will focus on developing a "Foodborne Viruses and Food Safety" course and food virus module for classroom education on the nation's leading cause of food illness Our proposed research will fill a major gap in our understanding of the ecology and survival of foodborne enteric viruses in fresh produce. This knowledge is needed to make possible the development and extension of effective preventive innovations. This project will also result in the development of practical ways to inactivate these viruses with approved technologies and use our existing industry network to ensure consumer food safety. In the long-term, our effort will improve food safety and public health.
<p>PROGRESS: 2011/09 TO 2012/08<br/>OUTPUTS: Fresh produce is a major vehicle for the transmission of human norovirus (NoV) because it is easily contaminated during both pre- and postharvest stages. The overall goal of this project is to determine the mechanism of viral contamination in fresh produce and to develop novel strategies to control foodborne viruses in fresh produce. (1) Internalization and dissemination of human norovirus and animal caliciviruses in hydroponically grown romaine lettuce. The roots of romaine lettuce growing in hydroponic feed water were inoculated with a human NoV or animal caliciviruses (Tulane virus [TV] and murine norovirus [MNV-1]), and plants were allowed to grow for 2 weeks. It was found that human NoV and animal caliciviruses attached tightly to the roots, became internalized via roots, and efficiently disseminated to the shoots and
leaves of the lettuce. (2) High pressure (HPP) inactivation of human NoV, it surrogates, and other enteric foodborne viruses. We have systematically investigated the effectiveness of HPP on inactivating enteric foodborne viruses in aqueous medium and fresh produce. We demonstrated that HPP treatment at 400 MPa for 2 min is capable of inactivating most viruses including MNV-1, TV, hepatitis A virus, and rotavirus. More than a 5-log virus reduction was achieved in all tested fresh produce (lettuce, cabbage, strawberry, and blueberry) under these conditions. However, pressure, pH, temperature, and salts conditions affect the effectiveness of the viral inactivation. In addition, food matrix can provide protective effects for virus inactivation. Using human NoV virus-like particles (VLPs) as models, we also demonstrated that HPP disrupted the structural and functional integrity of viral
capsid and disrupted the binding of VLPs to histo-blood group antigen (HBGA) receptors. (3) Novel interventions to remove viruses from fresh vegetables, fruits, tableware and food preparation utensils. We found that a panel of surfactants, including sodium dodecyl sulfate (SDS), Nonidet P-40 (NP-40), Triton X-100, and polysorbates, significantly enhanced the removal of enteric foodborne viruses from fresh fruits and vegetables. Moreover, a reduction of approximately 3 logs was observed in all the tested fresh produce after sanitization with a solution containing a combination of 50 ppm of each surfactant and 200 ppm of chlorine. This novel strategy (combination of surfactants and sanitizers) may be practical for removing surface viruses and thus enhance food safety and public health. (4) Education and extension. We have developed two courses addressing foodborne viruses including "Food
Safety and Public Health" and "Advanced Food Microbiology-Food Virology". We have published these results in high-impact journals including Applied and Environmental Microbiology, and PLOS one. We have presented and disseminated these findings in Institute of Food Technologists (IFT) and International Association for Food Protection (IAFP) meetings. We have also integrated these findings into extension and outreach programs including Center for Food Safety conference, and Advanced CIP Cleaning and Sanitation program. PARTICIPANTS: Jianrong Li (The Ohio State University), Lee-ann Jaykus (North Carolina State University), Ken Lee (The Ohio State University), Doug Doohan (The Ohio State University), Xi Jiang (University Of Cincinnati), Haiqiang Chen (University of Delaware), Uribe, Roberto (Kent State University). TARGET AUDIENCES: Professionals in food safety, food microbiology, food
virology, farmers, growers, fresh produce processors, county food safety extension agents. PROJECT MODIFICATIONS: Not relevant to this project.
<p>PROGRESS: 2010/09/01 TO 2011/08/31<br/>OUTPUTS: We have made significant progress on this project. (1) Enhanced removal of a human norovirus surrogate from fresh vegetables and fruits by a combination of surfactants and sanitizers. We found that a panel of surfactants, including sodium dodecyl sulfate (SDS), Nonidet P-40 (NP-40), Triton X-100, and polysorbates, significantly enhanced the removal of murine norovirus (MNV-1) from fresh fruits and vegetables. While chlorine solution (200 ppm) gave less than 1.2-log virus reductions in tested fresh produce, a solution containing 50 ppm of surfactant was able to achieve a 3-log virus reduction in strawberries and an approximately 2-log virus reduction in lettuce, cabbage, and raspberries. Moreover, a reduction of approximately 3 logs was observed in all the tested fresh produce after sanitization with a solution containing
a combination of 50 ppm of each surfactant and 200 ppm of chlorine. (2) Inactivation of a human norovirus surrogate in fresh produce by high-pressure processing (HPP). We have systematically investigated the effectiveness of HPP on inactivating murine norovirus (MNV-1) in aqueous medium and fresh produce. We demonstrated that more than a 5-log virus reduction was achieved in all tested fresh produce (lettuce, cabbage, strawberry, and blueberry) when it was pressurized at 400 MPa for 2 min at 4 degrees. We found that pressure, pH, temperature, and food matrix affected the virus survival in foods. MNV-1 was more effectively inactivated at 4 degrees than at 20 degrees in both medium and fresh produce. MNV-1 was also more sensitive to HPP at neutral pH than at acidic pH. We further demonstrated that disruption of viral capsid structure, but not degradation of viral genomic RNA, is the
primary mechanism of virus inactivation by HPP. (3) Inactivation of a human norovirus surrogate (MNV-1) by gamma irradiation. We demonstrated that MNV-1 was resistant to gamma irradiation. Only a 1.7- to 2.4-log virus reduction in fresh produce at the dose of 5.6 kGy was observed. We further demonstrated that gamma irradiation disrupted virion structure and degraded viral proteins and genomic RNA, which resulted in virus inactivation. Overall, our results suggest that viruses are much more resistant to irradiation than bacterial pathogens. (4) Electron-beam inactivation of a norovirus surrogate in fresh produce and model systems. We found that e-beam at 2 kGy provided less than a 1-log reduction of murine norovirus in PBS and DMEM. Irradiation of inoculated cabbage showed up to a 1-log reduction at 4 kGy, and less than a 3-log reduction at 12 kGy. On strawberries, less than a 1-log
reduction occurred at doses up to 6 kGy, with a maximum reduction of 2.21 log at 12 kGy. Overall, MNV-1 is highly resistant to e-beam. Ongoing experiments are aimed to determine the attachment and internalization of foodborne viruses in vegetables and fruits; to determine the efficiency of non-thermal processing technologies on virus inactivation in vegetables and fruits; and to integrate research findings to extension, outreach, and education programs. PARTICIPANTS: Jianrong Li (The Ohio State University), Lee-ann Jaykus (North Carolina State University), Ken Lee (The Ohio State University), Doug Doohan (The Ohio State University), Xi Jiang (University Of Cincinnati), Haiqiang Chen (University of Delaware), Uribe, Roberto (Kent State University) TARGET AUDIENCES: Professionals in food safety, food microbiology, food virology, farmers, growers, fresh produce processors, county food
safety extension agents PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.