UNDERSTANDING ENVIRONMENTAL RESERVOIRS AND PREVALENCE OF NOROVIRUS SURROGATES TO REDUCE IMPACT ON PUBLIC HEALTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0232564
• Project Start Date: Jan 1, 2013
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

Project Director
Gibson, KR.

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
Food Science

Non Technical Summary
An estimated 9.4 million episodes of foodborne illnesses due to known etiologies occur annually in the United States of which 5.45 million (58%) are caused by human norovirus (NoV). Food types most commonly associated with NoV outbreaks include ready to eat foods and in general, foods that are subject to minimal processing. Between 1990-2005, 64, 67, and 47% of outbreaks in the U.S. involving greens-based salads, fruits, and lettuce, respectively, were attributed to NoV, by far exceeding the contribution of all bacterial and protozoan foodborne pathogens. One of the major challenges the food industry faces today with respect to controlling NoV is the lack of 1) sensitive and specific detection methodologies for screening foods and water for virus contamination; and 2) a comprehensive understanding of environmental transmission. This research explores the characterization and use of novel NoV surrogates in research to understand transmission of NoV in fresh produce packinghouse environments.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4030	1101	70%
712	4050	1103	30%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4050 - Protozoa; 4030 - Viruses;

Field Of Science
1101 - Virology; 1103 - Other microbiology;

Keywords

norovirus

free living amoeba

fresh produce

virus surrogate

Goals / Objectives
The overall goal in this project is to obtain a better understanding of the transmission of human noroviruses (NoV) from the environment to food and subsequently to the public. Specific objectives: 1. Characterize novel surrogates for the study of human noroviruses. 2. Investigate environmental reservoirs for the transmission human norovirus surrogates via fresh produce. From this project, it is expected that 1) better NoV surrogates will be identified and utilized for the development of mitigation strategies to control NoV in our food systems and 2) the interaction of NoV with fresh produce via association with produce "microflora" will be established.

Project Methods
Virus [Tulane virus (TV), porcine sapovirus (PoSV)] survival at 37, 56, and 72 degrees C will be evaluated over various time points up to 5 days. Virus stability at pH 2, 3, 7, 9, and 10 will be evaluated at room temperature (RT) and 37 degrees C over various time points up to 3 hrs. Virus stability when exposed to 3 different concentrations each of peroxide, bleach, and ethanol will be evaluated at room temperature over various time points up to 1 hour. Virus survival in soil and water matrices will be evaluated at various time periods, temperatures, and levels of relative humidity. Virus persistence in surface water and groundwater will be determined at 4 degrees C and RT by inoculating the water samples at 105 PFU/ml. The samples will be incubated in the dark with continuous mixing for up to 5 weeks. Periodically, 1.5 ml subsamples will be removed and analyzed for virus infectivity by plaque assay using the corresponding host cell line. For virus survival in soil matrices, various types of soil will be collected, characterized (e.g., nutrients, metal oxides, conductivity, saturation pH, etc.), and inoculated with prepared virus surrogates at 105 PFU/g in a total of 5 g. The soil samples will be incubated for up to 8 weeks at optimal temperatures for growing leafy greens (i.e. 15 to 18 degrees C) as well as at 4, 10 and 25 degrees C. At each predetermined time point, subsamples will be removed and organic solvent extraction of viruses from the soil matrix will be applied. For all experiments the samples will be analyzed for virus infectivity by plaque assay. Obj 2: Free-living amoebae (FLA) and virus interactions will be evaluated. For co-culture of amoebae with NoV surrogates, experiments will be performed in suspension with a multiplicity of infection [MOI] of 0.33. The amoebae - virus suspensions will be incubated for one hour at 30 degrees C followed by centrifugation to pellet amoebic cells. The pellet will then be washed with PBS in order to remove unabsorbed viruses and maintained at 30 degrees C for up to 6 days. During this 6-day incubation, samples will be taken at 0, 6, 24, 48, 72, 96, 120, and 144 hours post-infection to determine virus titer within amoebae as well as cell-free supernatant. A plaque assay technique will be used to detect infectious virus particles. The transfer efficiency of FLA (with and without viruses) in water and on various surfaces to fresh produce will be determined. For transfer of FLA in water to fresh produce (i.e., leafy greens), experiments will be conducted with both ATCC and a subsample of wild-type Acanthamoebae spp. High and low level contamination experiments for each FLA and produce type will be repeated in triplicate. For transfer of FLA from contaminated surfaces to fresh produce, experiments will be conducted with both ATCC and a subsample of wild-type type FLA. For each FLA, 5 by 5 cm coupons of stainless steel or rubber conveyor belt material will be inoculated with high (106) or low (103) levels of FLA. High and low level contamination experiments for each FLA and produce type will be repeated 3 times.

Progress 01/01/13 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?From January 2013 to July 2017, I trained and graduated 4 M.S. students whose research was under the scope of this project. I am currently training 2 M.S. and 2 Ph.D. students whose work will also be related to this project. I also trained an Honors undergraduate student whose research focused on the characterization of microbial contamination on single-user mobile devices which would be considered environmental surfaces. How have the results been disseminated to communities of interest?Results have been dessiminated via publications, presentations, and student theses. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Through research publications and presentations, we have been able to show that hNoV and its surrogates behave differently under the influence of various physiochemical conditions. For instance, we found that hNoV surrogates interact differently with free-living amoeba (FLA) with MNV readily associating while FCV did not associate. This interaction may indicate that hNoVs will readily associate with FLA since MNV and hNoVs are more closely related. We also showed that FLA in association with MNV can readily transfer to fresh produce which indicates an other potential route of transmission. Within this research project, we also added to the literature for the characterization of Tulane virus (TuV)--a novel hNoV surrogate. Our research indicates that TuV on a surface is more resistant to chlorine inactivation than previously published results for inactivation in solution. In addition, we published the first study demonstrating the persistence of TuV in environmental (ground water and surface water) waters and on fomite surfaces. We also began to characterize the properties of Aichivirus A (AiV)--a frank human pathogen that is cultivable and can serve as a surrogate for human enteric viruses in general. Research from this project indicates that AiV can survive on various food contact surfaces for at least 14 days. With the surface type did not impact AiV survival, the relative humidity (RH) was found to be crucial with a 2 log difference in reduction over 14 days at 60% and 90% RH with greater survival at 90% RH. An additional research question that was undertaken within this project was to identify if hNoV genotypes impacted bleach inactivation kinetics in solution. Following 5 min exposure at 100 and 150ppm, GII.4 Sydney and New Orleans, GII.7, and GI.1 showed no change compared to untreated virus while a less than 1-log reduction was observed for GI.5 and GI.6. Conversely, GII.3, GII.6, GII.13, and GII.16 displayed complete inactivation (3 to 5-logs) at both 100 and 150ppm after 1 min exposure. An hNoV genotype dependent efficacy of bleach inactivation is important for practical application of inactivation research. Studies using only one or a limited number of genotypes might not convey the full spectrum of hNoV susceptibility or resistance. Under a similar scope with respect to inactivation/removal, another research project showed that non-antibacterial foaming soaps remove bacteria significantly better than our virus surrogate, MS2 bacteriophage. In addition, we found that study participants wash their hands approximately 5 seconds longer with gel-based soaps when compared to foaming soaps. This is important to understanding that the right tools will aid in the control of norovirus transmission. Another overall aim of this research was to allow for a better understanding of the fundamental importance of commensal microbiota--in a given environment--on hNoV survival and transmission. We hypothesize that hNoV interacts with the microbiome present in leafy green production systems through adsorption to the extracellular polymeric substances (EPS), lipopolysaccharide (LPS), and/or peptidoglycan (PG) of bacteria as a whole cell or as individual components thus aiding in the persistence and transmission of hNoV. Our data show that both hNoV and its surrogates rapidly associate with whole cell bacteria within 10 min with complete virus association within one hour. However, it seems the association with bacteria decreases the persistence of viruses under exposure to elevated temperatures (>37°C). Meanwhile, hNoV surrogate inactivation with bleach on stainless steel surfaces is dependent on the bacteria - surrogate virus combination with Gram positive bacteria such as B. cereus decreasing persistence.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Turnage NL, Gibson KE. Sampling methods for recovery of human enteric viruses from environmental surfaces Journal of Virological Methods. 248: 31-38. 2017.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Deng W, Gibson KE. Interactions of microorganisms within leafy green phyllospheres: Where do human noroviruses fit in? International Journal of Food Microbiology. 258: 28-37. 2017.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Turnage NL (2017). Persistence of Enteric Viruses on Surfaces Under Varying Environmental Conditions. M.S. Thesis. University of Arkansas: U.S.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Yu H, Gibson KE, Wright KG, Neal JA, Sirsat SA. Food safety and food quality perceptions of farmers market consumers in the United States Food Control. 79: 266-271. 2017.
• Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Conover DM. Comparative Efficacy of Foaming and Non-foaming Handsoap in reduction of Microorganisms in Handwashing. M.S. Thesis. University of Arkansas: U.S.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: International Association for Food Protection, Tampa, FL July 2017 Poster Presentation: Efficacy of Hypochlorite Bleach Treatment on Different Human Norovirus Genotypes Authors: Flor Maes, Genesis Chavez Reyes, Giselle Almeida, Kristen E. Gibson
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: International Association for Food Protection, Tampa, FL July 2017 Poster Presentation: Rapid Association of Enteric Viruses with Whole Cell Bacteria in Suspension Authors: Giselle Almeida and Kristen E. Gibson
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: International Association for Food Protection, Tampa, FL July 2017 Poster Presentation: Thermal Stability of Viruses in Coculture with Enterobacter cloacae Authors: Kristen E. Gibson and Giselle Almeida
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: International Association for Food Protection, Tampa, FL July 2017 Poster Presentation: Optimization of Virus Recovery from Non-porous Surfaces with Application in Environmental Persistence Studies Authors: Nicole Turnage and Kristen E. Gibson

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:I was invited to give presentations to both the Mid-Continental Association for Food and Drug Officials and the FDA Food/Feed Rapid Response Team on the topic of preventtion and control of human noroviruses. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I graduated one MS student that was working on a handwashing project using a norovirus surrogate though not immediately relevant to the project objectives. She published 3 papers on this topic. My PhD student was also awarded a fellowship from NoroCORE for her proposal on norovirus and surrogate interactions with bacteris present in the environment. She will be funded on this fellowship until May 2017. I have also been able to receive additional funding based on the data generated by my students. I have recruited another PhD student that started Fall 2016, and she is working on the interactions of human noroviruses and its surrogates with various components present in the leafy green phyllosphere and aspects of viral inactivaiton and/or persistence. How have the results been disseminated to communities of interest?Results have been dessiminated via publications, presentations, and student theses. What do you plan to do during the next reporting period to accomplish the goals?I would like to continue to investigate free-living protozoa (i.e. ciliates) interactions with enteric viruses to add to our work with free-living amoeba that was reported in the previous reporting period. In addition, I would like to investigate these associations using human noroviruses (HuNoV) and HuNoV virus like particles (VLPs) as opposed to just cultivable viral surrogates. I have been working on finding additional funding sources to do this work. We would also like to continue to investigate the environmental persistence of Tulane virus and other surrogates under a variety of conditions specfically on fomite surfaces relevant to the food industry and other important settings such as long-term care facilities and child care centers. We also acquired a new surrogate in our lab, Aichivirus A, and we plan to investigate the properties of this virus similar to the research we have completed with murine norovirus and Tulane virus. We have begun looking at this surrogate though the reserach is not completed. We will conitnue to look at virus-microbe interactions and determine the impact on persistence of the viruses under various conditions as well as the possibility that these interactions impact infectivity.

Impacts
What was accomplished under these goals? One of my MS students who graduated in May 2016 did her thesis on handwashing and the impact of different soap types. Her reserach showed that foaming soaps remove bacteria significnatly better than our virus surrogate, MS2 bacteriophage. In addition, we found that study participants wash their hands approximately 5 seconds longer with gel-based soaps when compared to foaming soaps. This is important to understanding that the right tools will aid in the control of norovirus transmission. My current PhD student has focused on optimization of recovery of viruses from various food contact surfaces. She is currently running experiemtns to determine the impact of temperature (refrigeration and RT) and relative humidity combinations on the persistence of viurs surrogates on different surfaces. The main conclusion from her method optimization is lack of standardization across publications and no reporting of recovery efficiencies to allow for comparison across studies. We have also been characterizing the histo-blood group antigen binding patterns of bacterial isolates that we have identified as present in the leafy green phyllosphere and/or the human gut as well as isolates we recovered from leafy greens sold at retail. By doing this we are able to understand potential interactions between human noroviruses and this specific bacteria. We have also optimized methods for isolation of bacterial LPS, PG, and EPS as we hypothesize that the viruses may be interacted with specific cell components as opposed to just the whole cell. On this front we have used TEM to visualize the virus association with whole cell bacteria. Last, we have conducted experiments on the impact of virus-bacteria association with respect to persistence at various temperatures. There does appear to be an effect of this association which allows the virus to persist for longer periods, but research is still ongoing.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Conover DM, Gibson KE. A review of methods for the evaluation of handwashing efficacy Food Control. 63:53-64. 2016.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Arthur SE, Gibson KE. Environmental persistence of Tulane virusa surrogate for human norovirus Canadian Journal of Microbiology. 62:449-454. 2016.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Conover DM, Gibson KE. Comparison of two plain soap types for removal of bacteria and viruses from hands with specific focus on food service environments Food Control. 69:141-146. 2016.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Almeida G, Gibson KE. Evaluation of a recirculating dipper well combined with ozone sanitizer for control of foodborne pathogens in food service operations Journal of Food Protection. 79:1537-1548. 2016.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Conover DM, Gibson KE. Impact of soap typefoaming vs. gel-basedon handwashing time Food Control. 73:878-882. 2017.
• Type: Book Chapters Status: Published Year Published: 2016 Citation: Gibson KE and Borchardt MA. 2016 Methods for Virus Recovery in Water pp. 277-301 In: Cannon, JL and Goyal SM (eds), Viruses in Food, 2nd edition. Springer US.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: University Council on Water Resources/National Institutes for Water Resources, Pensacola Beach, FL June 2016 Oral Presentation: Role of Freshwater Sediments in the Survival and Transport of Human Pathogens in the Environment Authors: Kristen E. Gibson

Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems:We have added Aichi virus A to this project due to the issues we have had with working with porcine sapovirus. What opportunities for training and professional development has the project provided?I have graduated two MS students that have worked on understanding the persistence and control of human norovirus surrogates. I have a current MS student that is working on a handwashing project using a norovirus surrogate though not immediately relevant to the project objectives. I have also been able to receive additional funding based on the data generated by my students. How have the results been disseminated to communities of interest?Results have been dessiminated via publications, presentations, and student theses. What do you plan to do during the next reporting period to accomplish the goals?I would like to continue to investigate free-living protozoa (i.e. ciliates) interactions with enteric viruses to add to our work with FLA. In addition, I would like to investigate these associations using human noroviruses (HuNoV) and HuNoV virus like particles (VLPs) as opposed to just cultivable viral surrogates. We would also like to continue to investigate the environmental persistence of Tulane virus and other surrogates under a variety of conditions specfically on fomite surfaces relevant to the food industry and other important settings such as long-term care facilities and child care centers. Last, we acquired a new surrogate in our lab, Aichivirus A, and we plan to investigate the properties of this virus similar to the research we have completed with murine norovirus and Tulane virus.

Impacts
What was accomplished under these goals? One of my MS students working on the interactions of viruses with free-living amoeba (FLA) graduated in December 2014, and from her research, she has published two peer-reviewed research publications on these interactions. One showed that murine norovirus readily interacts and associates with Acanthamoeba spp. while feline calicivirus shows no association. The other paper indicated that FLA in produce wash water can transfer to both loose leaf lettuce and tomatoes, and if associated with pathogens then this could be a food safety risk. My other MS students working on this research graduated in May 2015. He has one peer-reviewed publication that characterizing the physicochemial properties of Tulane virus, and his work corroborates previously published results, but also provides new information. For instance, we found that Tulane virus on a surface is more resistant to chlorine inactivation than previously published results for inactivation in solution. My student has another manuscript recently accepted (December 2015) that characterized Tulane virus stability in different water sources as well as on two surface types. This will be the first study to provide any data on this area of research.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Gibson KE, Almeida G, Conover D, Ghosh A, Arthur SE, Hsueh TY, Jackson J, Smith, L. Evaluation of Pathogen Survival in Fresh Water Sediments and Implications for Irrigation Water Quality 2015. In: IAFP 2015 Abstracts, July 25-28 Portland, OR. Journal of Food Protection. Abstract Nr P1=164. In Press.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wright K, Sirsat SA, Neal JA, Gibson KE. Growth of local food systems: a review of potential food safety implications. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources. 10:1-13. 2015.
• Type: Book Chapters Status: Published Year Published: 2015 Citation: Gibson KE. 2015 "Tracking Pathogens in the Environment: Applications to Fresh Produce Production" pp. 17-35 In: Ricke SC, Donaldson JR, Phillips CA (eds), Food Safety: Emerging Issues, Technologies and Systems. Elsevier US.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Hsueh TY, Gibson KE. Investigation of human norovirus surrogate interactions with Acanthamoeba spp. Applied and Environmental Microbiology. 81: 4005-4013. 2015.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Hsueh TY, Gibson KE. Transfer of Acanthamoeba spp. associated with viruses to fresh produce from water and environmental surfaces Letters in Applied Microbiology. 61:192-198. 2015
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Arthur SE, Gibson KE. Physicochemical stability profile of Tulane virusa human norovirus surrogate Journal of Applied Microbiology. 119:828-875. 2015.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: One of the proposed surrogates, porcine sapovirus, has been difficult to work with, but we are still making progress on the objectives though with some modifications. What opportunities for training and professional development has the project provided? I am currently supporting one masters student who is working on completion of Objective 1 for this M.S. thesis in the Cellular and Molecular Biology Program. In addition, an intern from Zamorano Agricultural University in Honduras studied in my lab in Spring 2014, and he learned methods for working with norovirus surrogates. 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? The gradute student working on Objective 1 will complete his research by May 2014. We also plan to submit proposals for future work that builds on Objective 2 and my first graduate student's research.

Impacts
What was accomplished under these goals? One of my students is graduating December 2014, and she has completed parts of the objective related to investigating environmental reservoirs of human noroviruses. She presented her work at the 2014 International Association for Food Protection (IAFP) Annual Meeting iN Indianapolis, IN. In addition, we submitted a manuscript to Applied and Environmental Microbiology which is currently under revision. My student also was awarded the Food Science Graduate Student award for 2014 based on her research in this area as well as her academic achievements. I hired an additional graduate student in Fall 2013, and he is working on primarily Objective 1. He presented his research at the local state chapter meeting of IAFP (Arkansas Association for Food Protection) and won first prize. In addition he won the graduate student research competitation in Food Science here at the University of Arkansas. Last, he wrote a review on methods used for evaluating the thermal stability of enteric virus surrogates, and this was accepted for publication in Food and Environmental Virology

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Hsueh TY and Gibson KE (2014). "Free-living amoeba as reservoirs for the transmission of norovirus." IAFP Abstracts: Proceedings of the 2014 International Association for Food Protection Annual Meeting in Indianapolis, IN. P2-166
• Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2014 Citation: Hsueh TY (2014). Acanthamoeba as reservoirs for transmission of norovirus. M.S. Thesis. University of Arkansas: U.S.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Arthur SE and Gibson KE (2015). Comparison of Methods for Evaluating the Thermal Stability of Human Enteric Viruses. Food and Environmental Virology. Accepted.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? I have hired 2 masters students to work on the objectives that have been laid out. These students will be trained on virus culture and detection methods as well as mentored by Dr. Gibson during their program of study. 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? The graduate student working on Objective 2 will continue collecting data on the interactions between amoeba and viruses as well as determine the role these interactions may play in contamination of fresh produce. In additon, the student will present findings at the International Association for Food Protection Meeting in July 2014. Last, the student will complete her thesis work in Fall 2014 which will include 2 publications related to Objective 2. The graduate student working on Objective 1 will begin collecting data on the environmental stability of novel norovirus surrogates in Spring 2014, and this will continue through the end of 2014.

Impacts
What was accomplished under these goals? I have one graduate student that is generating data on the interaction of norovirus surrogates with Acanthamoeba spp. (Objective 2). The student has repeated initial tissue culture experiments in duplicate and has shown a significant difference in interactions with amoeba between murine norovirus and feline calicivirus, both norovirus surrogates. These results will be used as part of the student's masters thesis and also will be submitted for presentation at the International Association for Food Protection Meeting in 2014. In addition, these results will be used as preliminary data for submission of grants to the AFRI Food Safety Program as well as to the Center for Produce Safety. WIth respect to Objective 1, I have hired a masters student to characterize novel surrogates for the study of human noroviruses. He will begin his research in Spring 2014.

Publications