Food-borne Enteric Calicivirus Interactions with Damaged, Stressed and Pathogen-infected Leafy Greens

<p>NON-TECHNICAL SUMMARY:<br/> Norovirus (NoV) contamination of leafy greens is a major cause of foodborne gastroenteritis outbreaks in the US. Preliminary experiments by Dr Wang suggest that a cultivable norovirus surrogate, the enteric calicivirus, porcine sapovirus (SaV) not only attaches to the surface of lettuce leaves, but also internalizes into leaves and disseminates systemically within contaminated plants. Based on these results, we hypothesize that specific sugar-related surface interactions, resembling those in the mammalian host, occur between NoV/SaV and leafy greens. These interactions and conditions that perturb plant health may enhance the attachment/dissemination/retention of NoV in leafy greens. To test this hypothesis, lettuce and spinach will be subjected to various treatments that alter surface sugar residues or weaken plant defense responses. We will
examine treated plants for altered attachment/dissemination/retention of the NoV surrogates. The treatments include mechanical damage, abiotic stresses (heat and flooding), infections with plant pathogens (viruses and bacteria) and treatments that enhance plant anti-microbial defense. Because NoVs are uncultivable, stressed plants will be exposed to two types of NoV surrogates, virus-like particles (VLP) of human NoV and infectious porcine SaV. The surface attachment/uptake/retention of SaV/VLPs will be investigated using detached leaves. Their internalization and systemic dissemination will be followed in intact plants. Successful completion of the proposed studies will advance our understanding of the interactions between NoV/SaV and leafy greens, leading to improved preventive measures to reduce or eliminate NoV-related outbreaks of foodborne illness and enhance public health.
<p>APPROACH:<br/> Lettuce and spinach will be subjected to various treatments that alter surface sugar residues or weaken plant defense responses. We will examine treated plants for altered attachment/dissemination/retention of the NoV surrogates. The treatments include mechanical damage, abiotic stresses (heat and flooding), infections with plant pathogens (viruses and bacteria) and treatments that enhance plant anti-microbial defense. The studies using plants infected with other plant pathogens and also the studies of the use of the plant activator ASM are novel. Because NoVs are uncultivable, stressed plants will be exposed to two types of NoV surrogates, virus-like particles (VLP) of human NoV and infectious porcine SaV. The mean differences among experimental groups (within day) and among sample dates (within group) will be compared using analysis of variance (ANOVA)
and separated using Fisher's Protected Least Significant Difference (LSD) test. Differences between groups for the percentage of plants (incidence) with systemic dissemination of VLPs/SaVs (non-inoculated interior leaves positive for VLPs/SaVs) will be analyzed by Fisher Exact test. The relationship between sugar levels and SaV titers or number of VLPs will be investigated. The SaV titers and number of VLPs will be plotted against sugar levels and regression techniques will be used to determine optimal predictive models. All statistical tests will be performed using SAS (version 9.2). Data is considered to be statistically significantly different if the P value is <p>PROGRESS: 2012/02 TO 2013/01<br/>OUTPUTS: Human norovirus (NoV) is currently the leading cause of foodborne disease. The virus does not grow in cell culture and hence its survival in leafy greens under different environmental stressors cannot be determined in vitro. We used a cultivable enteric calicivirus, porcine sapovirus (SaV), as a surrogate to study the uptake and dissemination of NoV in lettuce and spinach under different environmental stressors. Green house experiments were conducted whereby the plants were subjected to either physical (leaves cracked in the middle) or heat stresses (36 C degree for two days) before being inoculated with SaV. The virus was inoculated through the roots and the roots and leaves tested after exterior disinfection with chlorine (1000 ppm for 5 min). Sampling and RNA extraction followed by SaV-specific real-time reverse
transcription (RT)-PCR were performed on post-inoculation day (PID) 1, 3, 7, 10 and 14. Our preliminary results showed that the SaV RNA was detected inside the roots of non-stressed control plants and physically as well as heat stressed plants. In general, the virus was detected inside the roots at a higher titer in physically and heat stressed spinach plants than that in lettuce plants. However, by PID 14, SaV had similar RNA titers in both plants under both stressors. The viral RNA also internalized inside lettuce and spinach leaves under both environmental stressors and persisted until PID 14, like in the non-stressed control plants. Compared to non-stressed control plants, SaV had higher titers in lettuce leaves at PID1 and 3 under heat and physical stress, but by PID 14 the titer was similar under either environmental stressor. In leaves of spinach plants, the viral RNA titer
followed a similar trend to the controls under both environmental stressors. SaV RNA titer was higher in spinach leaves as compared to lettuce under both environmental stressors. Many plant viruses have been reported to infect lettuce, including cucumber mosaic virus, turnip mosaic virus, tomato spotted wilt virus and tomato bushy stunt virus. However, we were unable to get consistent infection by these viruses. We infected lettuce with the bacteria (Xanthomonas campestris pv. vitians s. 701a) and compared SaV contamination and persistence in the plant bacterial-infected and control lettuce. We found that the bacterial-infected lettuce retained SaV slightly longer than in healthy lettuce. On the other hand, spinach plants infected with cucumber mosaic virus showed similar duration of SaV retention as healthy spinach. We are in the process of repeat the above experiments and make
conclusions. PARTICIPANTS: Dr. Qiuhong Wang, PhD (20% effort), PD is a Research Scientist, Adjunct Assistant Professor in Food Animal Health Research Program/OARDC and Veterinary Preventive Medicine. She has overall responsibility with the co-PD for the proposed project including the experimental design, generation of reports, manuscripts, etc. Other responsibilities include the supervision and interpretation of the experiments, supervision of virology assays and coordination of research efforts. E-mail: Wang.655@osu.edu Dr. Feng Qu, PhD (15% effort), co-PD is an Assistant Professor in Plant Pathology/OARDC. He supervises and assists in the conduct of all the plant (lettuce and spinach) experiments described and in the data analysis and interpretation. E-mail: qu.28@osu.edu Dr. Linda J. Saif, PhD (5% effort), Professor in Food Animal Health Research Program/OARDC and Veterinary
Preventive Medicine. She provides the culturable porcine SaV, which was used as the surrogate for human NoV, assists with the proposed project including providing advice and assistance as needed on the virology assays. E-mail: Saif.2@osu.edu Dr. Sally Miller, PhD (3% effort), Professor and co-investigator in Plant Pathology/OARDC. She advises or assists students or laboratory personnel in the plant production and phytopathology aspects of this project. E-mail: Miller.769@osu.edu Dr. Melanie Lewis Ivey, PhD (5% effort), is a Research Scientist and co-investigator in Plant Pathology/OARDC. She works with the students and lab personnel to assist with the plant production and phytopathology aspects of this project as well as arrangement of growth chambers, as well as the diagnostic aspects and data analysis. E-mail: Ivey.14@osu.edu Dr. Malak Essseili, Post-doctoral researcher (50% effort) is
a post-doctoral researcher in Food Animal Health Research Program/OARDC. She performed the study of uptake and dissemination of SaV in lettuce and spinach under physical and heat stressors. She performed data analysis and interpretation and wrote the report. E-mail: esseili.1@osu.edu Mrs. Ashlina Chin, BS, Graduate Research Associate, MS candidate in Plant Pathology/OARDC, performed the study of uptake and dissemination of SaV in lettuce and spinach under plant bacterial and virus infection-stressors. She performed corresponding data analysis and interpretation. E-mail: chin.131@buckeyemail.osu.edu Mr. Zhenwen zhang, BS, Research Assistant (50% effort)in Food Animal Health Research Program/OARDC by May, 2012, assisted Dr. Esseili with experiments. E-mail: zhang.774@osu.edu TARGET AUDIENCES: Food safety researchers, virologist, public health state and federal agencies involved in food
safety, vegetable producers and processors, consumers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
<p>PROGRESS: 2011/02/01 TO 2012/01/31<br/>OUTPUTS: Since human norovirus (NoV) is non-cultivable, we used a cultivable enteric calicivirus, porcine sapovirus (SaV), as a surrogate. To study the uptake and dissemination of SaV in lettuce exposed to physical damage, green house experiments were conducted whereby the outer leaves or roots of lettuce plants were damaged physically before being subjected to SaV treatment through: (1) swabbing the damaged outer leaves; (2) submerging the damaged roots; or (3) watering the soil of plants with damaged roots with a SaV solution and assaying for the virus in the leaves. For soil treated plants, sap was collected as it exuded from the cut stems and was tested for SaV RNA. Sampling on PID 0, 1, 2, 3, 5, 7, 14 and 21 and RNA extraction followed by SaV- real-time reverse transcription (RT)-PCR were performed. Our preliminary analysis
showed that SaV RNA persisted with higher titer on outer leaves that were damaged vs. control non-damaged leaves for at least 21days (market ready lettuce). However, the virus was not transferred from outer to inner leaves. When the roots of young lettuce plants were directly contaminated with SaV, the viral RNA persisted similarly for at least 21 days on the roots of damaged and control non-damaged plants and could often be transferred to leaves in both groups. When the soils of ready-to-harvest lettuce were contaminated with SaV, the virus persisted on the roots of damaged and control non-damaged plants for at least 21 days and was often detected in the sap of both groups. However, the virus was only detected for 2 days post soil inoculation in the leaves of control plants but was detected until day 5 in the leaves of plants with damaged roots. PARTICIPANTS: Individuals who worked on
this project: Dr. Qiuhong Wang (PI), Research Scientist, FAHRP, OARDC/The Ohio State University (OSU); Dr. Feng Qu (co-PI), Assistant Professor, Plant Pathology, OARDC/OSU; Dr Linda J Saif, Distinguished University Professor, FAHRP, OARDC/OSU; Dr. Malak Esseili, Post-doc, FAHRP, OSU; Zhenwen Zhang, Visiting Scholar, FAHRP, OSU; Ashlina Chin, graduate student, Plant Pathology, OSU; Dr. Sally Miller, Professor, Plant Pathology, OSU; Melanie Ivey, Research Associate and Graduate Student, Plant Pathology, OSU. TARGET AUDIENCES: Food safety researchers, virologists, public health state and federal agencies involved in food safety, vegetable producers and processors, consumers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.