THERMAL INACTIVATION OF NOROVIRUS IN OYSTER: QUANTIFYING THE RISKS ASSOCIATED WITH RESIDUAL VIRUSES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1029851
• Grant No.: 2023-67017-39184
• Cumulative Award Amt.: $617,396.00
• Proposal No.: 2022-09032
• Project Start Date: Mar 1, 2023
• Project End Date: Feb 28, 2027
• Grant Year: 2023
• Program Code: [A1332]- Food Safety and Defense

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
(N/A)

Non Technical Summary
Human norovirus is the leading cause of foodborne illnesses in the United States. It causes swelling of the stomach and intestines. Illnesses with norovirus occur when pathogen is ingested through the consumption of raw or undercooked food. Food commodities, such as oysters, have a remarkably higher risk of carrying the virus. Oysters live in water and obtain their food through filtering water, through which they also entrap microbes and food particles. Viruses are generally known to last long in oysters and are more tolerant than bacteria to partial cooking. Norovirus is one of the hardy pathogens that spread quickly in a community. Despite cooking being the cheapest and most effective method for protecting humans from illnesses, several norovirus outbreaks have been reported after eating cooked oysters, indicating high tolerance of norovirus to current cooking practices. The binding of viruses to each other or food particles are believed to protect them from cooking. Our interdisciplinary food virology, food engineering, and risk assessment team address this critical concern by studying the conditions that may lead to the survival of norovirus in oysters during the cooking practices (roasting, baking, and steaming) used by consumers and restaurants. Different forms of norovirus presence in oysters will be tested to understand their response to heat. Risks of consumption will be investigated using laboratory-driven data. The outcomes of this study aim to reduce the burden of illnesses associated with norovirus so consumers keep enjoying eating oysters.

Animal Health Component

70%

Research Effort Categories

Basic

30%

Applied

70%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
723	4030	3020	20%
723	4030	1101	20%
723	0811	1101	10%
501	0811	1101	10%
501	0811	3090	10%
723	7310	1101	30%

Knowledge Area
501 - New and Improved Food Processing Technologies; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
7310 - Experimental design and statistical methods; 4030 - Viruses; 0811 - Shellfish;

Field Of Science
1101 - Virology; 3090 - Sensory science (human senses); 3020 - Education;

Keywords

food safety

norovirus

oyster

public health

risk assessment

thermal processing

Goals / Objectives
The proposed research takes a risk-based approach to mitigate norovirus in heat-treated oysters. Human norovirus is the leading cause of foodborne diarrheal diseases worldwide. Our interdisciplinary team, with expertise in food virology, engineering, and quantitative risk analysis, aims to investigate the thermal inactivation of norovirus in buffered solutions, homogenized oyster tissues, and individual half-shell oysters following consumer and restaurant practices. To achieve our goals, we have established four objectives: 1) Identify current practices of cooking oysters at consumer and industry levels, 2) Determine the impact of dispersion and aggregation on thermal inactivation of norovirus, 3) Assess thermal inactivation of norovirus in oysters using common practices, i.e., steaming, baking, and roasting, and 4) Establish quantitative risk assessment models for norovirus in partially and fully cooked oysters. The outcome of this research will provide risk managers (e.g., policymakers, industry, restaurants) with practical knowledge on mitigating the risks associated with consuming cooked oysters.

Project Methods
Objective 1: Identify current practices of cooking oysters at consumer and industry levelsConsidering the diversity of cooking strategies, we will identify current preferred cooking methods (roasting, baking, and steaming) at consumer and industry levels through surveys of consumers (online/phone) and restaurant settings (phone/on-site). The interviews will be performed after ethical approval by the University of Florida Institutional Review Board (IRB). An online cross-sectional study of consumers and restaurant settings will be conducted with a quantitative and qualitative-based questionnaire using Qualtrics (Qualtrics, USA). Other information such as cooking ware, how the oysters are shucked and laid on the trays, and characteristics of trays will be recorded as well. Based on these data, we will implement the current processing parameters in our experimental design for developing virus inactivation and risk assessment models.Objective 2: Impact of dispersion and aggregation on thermal inactivation of norovirusPreliminary heat inactivation experiments will be performed using purified Tulane virus (TuV) and murine norovirus (MNV), two surrogates for human norovirus. The selected surrogate will then be used for the rest of the experiments across broader time and temperature combinations. We will generate virus-like particles for human norovirus and designated surrogate using Bac-to-Bac® TOPO® Expression System (Thermo Fisher Scientific) to assess aggregation and other structural changes during heat treatment. Aggregated or dispersed virus particles and their VLPs will be subjected to a range of heat treatments and then cooled down to 20°C. Virus survival will be tested with plaque assay (surrogate) and RNase RT-qPCR (surrogate and human norovirus). Characteristics of virus particles will be assessed with TEM, SDS-PAGE, and DLS on VLPs or a high-titer surrogate virus.Objective 3. Assess thermal inactivation of norovirus in oysters using common practices, i.e., steaming, baking, and roastingWe aim to simulate current consumer and restaurant-level cooking practices (Objective 1) under controlled conditions. Thermocouples will be used to assess the heat transfer dynamic in oyster tissues during cooking regimes to determine the thermal kinetics and diffusivity values. This will facilitate heat transfer and inactivation modeling over a wide range of cooking regimes. Oysters will be shucked and dissected to excise gills, digestive diverticula, and adductor muscles. The homogenized oysters will be thawed in the refrigerator. Some half-shell oyster samples, oysters will be shucked prior to the experiments. Norovirus or CSV particles in the forms of dispersed and aggregated will be used for inoculating tissue homogenates. The temperature will be monitored with thermocouples in virus-free samples. Following the thermal treatments, the homogenate will be cooled by placing the tube in iced water before the analysis. Half-shell oysters will be inoculated by injecting dispersed or aggregated virus particles into the digestive diverticula. After 15 min for attachment time, samples will be subjected to heat treatment. Type T thermocouples will be inserted through the shell into the digestive diverticula to target the most critical spot in oyster soft tissues and calculate the come-up time. The come-up time will be tested across other soft tissues of oysters. Cold spots will be identified through finite elemental analysis. Virus extraction follows FDA BAM protocol using the ultracentrifugation technique combined with the adsorption-elution technique or other alternative methods. Virus testing will follow plaque assay (surrogate) and RNase RT-qPCR (surrogate and human norovirus). Once suitable time-temperature combinations are identified that result in a desired thermal inactivation, non-inoculated samples will be heat treated similarly and subjected to Kramer shear force testing and consumer acceptance testing to evaluate the impact of cooking techniques on the quality attributes of oysters.Objective 4: Establish quantitative risk assessment models of norovirus in partially and fully cooked oystersThe risk assessment will model the prevalence and concentration of norovirus from harvest to consumption using current cooking practices (steaming, roasting, baking) in the baseline scenario. Temperature profiles and virus inactivation during cooking (norovirus, surrogate viruses) will be estimated from newly generated data in this project using linear or non-linear models as appropriate. Consumer risks will be estimated using cooked oyster meals as commonly served in the US. Based on project results, alternative cooking scenarios will be proposed to reduce exposure to norovirus in cooked oyster meals. Different published dose-response models will be evaluated for their impact on consumer risk estimates and risk reduction by alternative cooking practices.

Progress 03/01/24 to 02/28/25

Outputs
Target Audience:This project targets a diverse audience, including the general public, academics, the food industry, and public health agencies, all of whom play a critical role in enhancing public health through the adoption of safe food handling and cooking practices. Additionally, students and educators involved in this research are direct beneficiaries, as their participation aligns with the project's goals to address significant public health challenges, particularly regarding the safety of undercooked oysters--a high-risk food category identified by the CDC for human norovirus contamination. During the reporting period, the project focused on engaging the following key audiences: General Public: By disseminating findings through outreach activities and educational materials, the project empowers individuals to adopt safer cooking practices, reducing their risk of foodborne illnesses. Academics and Educators: Students and faculty participating in the research gained hands-on experience and advanced knowledge in food safety, contributing to workforce development in public health and food science. The project's updateshave been shared with scientific communities through technical posters and talks. Food Industry Professionals: The project surveyed over 100 restaurants in the United States (Objective 1) to collect valuable insights into current oyster cooking practices (baking, roasting, steaming) and identify gaps and opportunities for improving food safety measures. This information supports the development of risk modelsthat assess norovirus resistance to heat and identify effective time/temperature conditions for the safe cooking of oysters. Public Health Agencies: The findings aim to guide agencies in recommending evidence-based and risk-based policies and interventions to enhance food safety at a community and national level. By tailoring efforts toward these audiences, the project seeks to foster collaboration between researchers, industry practitioners, and policymakers to implement more effective food safety practices. This work not only advances scientific understanding of the response of norovirus to cooking in food but also ensures that actionable recommendations are communicated to those most directly impacted by foodborne risks. Changes/Problems:The survey focused narrowly on identifying oyster cooking techniques used by the public and restaurants, which led to unexpected challenges. In total, 23 online surveys were started, and 12 were fully completed. Of the 3,506 total phone records (which includes an online sample ported into the phone survey), 464 were disconnected or non-working numbers, and 334 establishments said they did not or no longer served oysters. In total, the UFSRC interviewers conducted 94 phone interviews, with the remainder gathered via Qualtrics, culminating in 106 completed surveys. As a result of this intricacy, the expenses involved in reaching the intended audience notably surgedcompared to the original estimate. Although the low response rate posed a challenge, the data gathered was adequate to create a robust foundation for fulfilling Objective 3. Furthermore, the consideration of murine norovirus alongside TuV and HuNoV was also removed from Objective 2 because of the complexities of the ongoing experiments. Tulane virus serves as an robust surrogate for HuNoV and its utilization in this project offer important insights into the efficacy of heat treatments for norovirus inactivation. The use of Dynamic Light Scattering (DLS) and Nanoflow Cytometry to measure the numbers and properties of virus particles (Objective 2) hasbeen hindered by virus concentration. Although ultracentrifuge-purified virus stocks are highly pure, achieving virus concentrations consistently above 9.5 log10PFU/ml has been challenging. Additionally, the 40 nm size of Tulane virus (TuV) is at the sensitivity threshold of the Nanoflow Cytometer, complicating efforts to accurately measure total virus particles andparticle-to-PFU ratio. Efforts are ongoing to improve instrument calibration and explore alternative tools to enhance the accuracy and reliability of these measurements. What opportunities for training and professional development has the project provided?This project has offered numerous opportunities for two undergraduate students, two MSc students, and onePh.D. studentto advance their academic and professional development, whether as master's or Ph.D. students or as interns gaining hands-on experience in scholarly research. Under the guidance of the PD, Co-PDs, and external members of graduate committees, mentees have been actively involved in various aspects of the project. Their contributions included conducting experimental research, maintaining cell lines, conducting surveys, data collection/analysis/reporting, collectively providing them with skills and knowledge in their respective fields and readiness for the workforce in agricultural sciences. Through the support of the grant, further training was provided for the PD to improve skills in microbial risk assessment: 1) Quantitative Microbial Risk Assessment Interdisciplinary Vehicle (QMRA IV) - Center for Advancing Microbial Risk Assessment (CAMRA) at Michigan State University, East Lansing, MI. Duration: 18-h online, June 1-30, and 44-h in-person, Jul 30-Aug 4, 2023. and 2) Modeling Food Safety and Animal Health Risks Using R - EpiX Analytics. Duration: 45-h of online synchronous/asynchronous course, May 8-26, 2023. The skills were incorporated into teachingand research efforts and to train mentees involved in the project.The PD serves as the instructor for both the lecture and laboratory sessions of the Food Microbiology course (FOS 4222/4222L/5225C), core courses in the food science curriculum designed for upper-division undergraduate and graduate students. The course content has been enhanced by incorporating and expanding modules on predictive modeling and microbial risk assessment. These improvements give students a deeper understanding of how models canpredict changes in microbial populations and how hazard and risk are applied to assess the likelihood of illnesses caused by foodborne pathogens. As aboard member, the PD actively participated as a speaker coordinator for the Florida Association for Food Protection (FAFP) Annual Education Conference (AEC) meetings in 2023 and 2024. Through these efforts, the PD led and co-led the organization of sessions featuring speakers from academia, industry, and governmental agencies. These meetings provided valuable opportunities for students involved in the project, enabling them to receive travel awards and present their research findings to a diverse audience. How have the results been disseminated to communities of interest?Data have been presented as poster and oral talks at local, state, and international annual meetings to reach out to audiences in academia, industry, and government (see Products). Experimental data from Objectives 1 and 2 are under preparation for publication in peer-reviewed journals. Two technical articles on the proper cooking of foodhave been submitted to the UF/IFAS EDIS (Electronic Data Information Source of UF/IFAS Extension) to reacha broader audience, including the general public. What do you plan to do during the next reporting period to accomplish the goals?Future work will focus on optimizing aggregation conditions using polyethylene glycol and peptides, studying thermal inactivation kinetics of aggregated versus dispersed virus populations, and applying these findings to norovirus surrogates in diverse environmental matrices.The objective 3 will continue to assess the effectiveness of cooking techniques following the conditions obtained from Obj 1.

Impacts
What was accomplished under these goals? Objective 1 of the project was achieved through a comprehensive survey of U.S.-based restaurants, focusing on their oyster cooking methods, including steaming, baking, and roasting. The University of Florida Survey Research Center (UFSRC) administered the survey as a fee-based service. Data were collected from seafood establishments using Standard Industrial Classification (SIC) Codes, employing phone surveys and the Qualtrics platform. The ethically approved and pre-tested questionnaire featured a mix of open- and close-ended questions targeting restaurant staff, including owners, managers, and chefs. Key topics explored included customer and serving volumes, oyster sourcing,commoncooking methods, preparation times and temperatures, and thermometer usage. This study sheds light on oyster cooking practices in U.S. restaurants. It provides valuable data to support the development of robust risk assessment models for pathogen inactivation, enhance food safety guidelines, and reduce health risks associated with oyster consumption. The experimental research in this project usesclinical specimens of human norovirus (HuNoV), when applicable, andTulane virus (TuV), a HuNoV surrogate. Tulane virus particles were purified from cell lysates via ultracentrifugation and quantified using plaque assays. For HuNoV, the lack of a reliable cultivation system necessitates the use of RT-qPCR as the gold standard for detection and quantification. To evaluate heat-induced virus inactivation, both TuV and HuNoV were analyzed using RT-qPCR following RNase treatment prior to RNA extraction. This method allows for the quantification of viral RNA derived from intact, presumably infectious viral particles. The combined approach provides critical insights into virus inactivation mechanisms and informs the development of effective intervention strategies.To support the overarching goal of this research project, preliminary experiments focused on establishing a particle-to-PFU (P:PFU) ratio for quantifying virus particles. The results of this study were disseminated as poster presentations at the 2024 International Association for Food Protection (IAFP) Annual Meeting, the 2024 Florida Association for Food Protection conference, and the UF Emerging Pathogens Institute (EPI) Research Day. Additionally, a manuscript based on this work is pending publicationatthejournalofFood and Environmental Virology. The Objective 2research efforts were focused on determining how dispersion and aggregation affect the thermal inactivation of norovirus. To explore virus aggregation, we induced aggregation using pH adjustment and ionic strength, with plans to include polyethylene glycol and peptides in future studies. Measuring the isoelectric point (pI) ofTulanevirus, a surrogate for norovirus, was critical due to the lack of reliable data. Zeta potential measurements using a Zetasizer Nano ZS were initially inconsistent, prompting several adjustments: filtering solutions, increasing virus concentration, and matching the sample and capillary buffer. Further refinement of software settings based on Malvern Technology recommendations resolved measurement issues.Preliminary work on Objective 3 has been initiated by studyingthe impact of organic loads on the thermal inactivation kinetics of TuV and assessing the survival of residual virus particles. Phosphate-buffered saline (PBS) served as the control matrix, andHuNoV GII-positive stool and oyster digestive diverticulum were used as virus-associated matrices. Control experiments using PBS to suspend TuV were conducted at 50-60°C for varying durations. Data from these experiments are currently under analysis. Preliminary findings indicate that RNase RT-qPCR data show less viral reduction compared to plaque assay results.Thissuggests that RNase RT-qPCR is sensitive enough to detect and quantify residual virus particles that are below the plaque assay's limit of detection. Current efforts are focused on testing the inactivation of TuV and human norovirus (HuNoV) in oyster digestive diverticulum and evaluating the recovery efficiency of the virus extraction method described in the FDA's Bacteriological Analytical Manual (BAM). This extraction technique was selected for its ability to recover virus particles while maintaining their infectivity; however, its variability in recovery efficiency and long processing times are noted challenges. Under Objective 3, a 3D scanning workflow for oyster shells has been established and refined using Blender, MeshLab, and SpaceClaim. Large, medium, and small PLA oyster prototypes were 3D-printed for preliminary heat transfer studies. Parallel experiments with real oysters were conducted to evaluate the feasibility of using 3D-printed models for heat transfer analysis. Upcoming experiments will involve inoculating oysters with Tulane virus TuV and HuNoV to study virus inactivation kinetics under various cooking conditions. Several dozen thermal experiments have been conducted to assess the thermal penetration of real oysters and printed models using various boundary conditions. These efforts continue to accurately describe and model the thermal penetration of oysters and connect the results to the thermal models. The 3D-printed oyster models are in the process of being subjected to three primary cooking methods, replicating typical culinary practices. Each cooking method is standardized for time and temperature, matching that of acommonrestaurant setting, as obtained from the survey conducted by Dr. Montazeri's team. Experiments will involve various cooking methods, including steaming, baking, and roasting. Each technique is being performed under controlled conditions, with consistent temperature settings (information from the survey) and durations to ensure reproducibility.T-type Thermocouples will be inserted into the 3D models at different points to record internal temperatures, adapting the methodology outlined in previous research for accurate thermal monitoring. The thermocouples were connected to a data logger (Graphtec GL 240) to collect data at regular intervals and will be recorded on a computer.Temperature data was systematically recorded throughout the cooking process for all three cooking methods. Simulations under various cooking conditions are being conducted to predict the temperature distribution within the oyster models over time. Experiments to test the boundary conditions in real-world scenarios are beingconductedto confirm the simulation models.?Ansys is a Finite Element Method (FEM) integrated with the key thermal properties such as thermal conductivity, Specific heat, and Thermal diffusivity obtained from the experimentations and literature values. This data and thermal model will be further refined as a reference for the prediction of norovirus inactivation.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Razieh Mirmahdi, Samantha Dicker, and Naim Montazeri. Unveiling the correlation between infectivity assay and RT-qPCR in norovirus detection using Tulane virus as a surrogate. IAFP Annual Meeting, Long Beach, CA. July 27-30, 2024 (poster).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Samantha Dicker, Razieh Mirmahdi, and Naim Montazeri. Impact of dissolved organic compounds on the thermal inactivation of norovirus. FAFPAEC, St. Petersburg, FL. May 14-16, 2024 (poster and short talk). FAFP Travel Award to S. Dicker.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Razieh Mirmahdi, Samantha Dicker, and Naim Montazeri. Unveiling the correlation between infectivity assay and RT-qPCR in norovirus detection using Tulane virus as a surrogate. FAFPAEC, St. Petersburg, FL. May 14-16, 2024 (poster and short talk). FAFP Travel Award to R. Mirmahdi.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Razieh Mirmahdi, Samantha Dicker, and Naim Montazeri. Unveiling the correlation between infectivity assay and RT-qPCR in norovirus detection using Tulane virus as a surrogate. Future of Food Forum, University of Florida, Gainesville, FL. Apr 3, 2024 (poster).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Razieh Mirmahdi, Samantha Dicker, and Naim Montazeri. Assessing the efficiency of RT-qPCR in distinguishing infectious from non-infectious norovirus using Tulane virus as a surrogate. Graduate Research Day (GSRD)  University of Florida, Gainesville, FL. April 2, 2024 (poster).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Razieh Mirmahdi, Samantha Dicker, and Naim Montazeri. Assessing the efficiency of RT-qPCR in distinguishing infectious from non-infectious norovirus using Tulane virus as a surrogate. Emerging Pathogens Institute Research Day, University of Florida, Gainesville, FL. Feb 15, 2024.
• Type: Other Journal Articles Status: Under Review Year Published: 2024 Citation: Razieh Sadat Mirmahdi, Samantha Dicker, Razieh Farzad, Andrew J. MacIntosh, Amarat Simonne, and Naim Montazeri. Ensuring Safe Oysters: Essential Handling, Preparing, and Cooking Practices. UF/IFAS EDIS.
• Type: Other Journal Articles Status: Under Review Year Published: 2024 Citation: Samantha Dicker, Razieh Sadat Mirmahdi, and Naim Montazeri. Cook It Right: A Guide to Safeguard Your Food Through Proper Cooking. UF/IFAS EDIS
• Type: Peer Reviewed Journal Articles Status: Awaiting Publication Year Published: 2025 Citation: Razieh Sadat Mirmahdi, Samantha L. Dicker, Nuradeen Garba Yusuf, and Naim Montazeri. Navigating uncertainties in RT-qPCR and plaque assay for infectivity assessment of norovirus. Food and Environmental Virology.

Progress 03/01/23 to 02/29/24

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two graduate students (one MS and one PhD) were hired to perform Objs1, 2, and 3 of the project. Training and protocol optimization/establishment. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Launching and completing the Obj1 survey. Performlaboratory-based experiments for the Obj1 and 2.

Impacts
What was accomplished under these goals? The IRB for Obj1 is approved for performing the behavioral survey. We are in the process of recruiting a company to perform the phone and online survey.

Publications