Human Noroviruses: Mechanisms of Attachment and Internalization in Leafy Greens

<p>NON-TECHNICAL SUMMARY:<br/> Human noroviruses (NoV) are the leading cause of foodborne illness in the US. NoVs bind to carbohydrates of lettuce leaf cell wall materials (CWM). Viral RNA is also transported through the roots into lettuce leaves. Knowledge about the mechanisms of NoV binding to leafy greens, how viral particles are transported from roots to leaves and if they retain infectivity is critical to develop technologies to control NoV contamination. Our hypotheses are: 1) NoV binding to lettuce carbohydrates is mediated through NoV histo-blood group antigen (HBGA)-binding pockets and binding may enhance virus persistence and facilitate entry into leaves; and 2) When contamination occurs through roots, enteric caliciviruses can be transported into leaves through the plant's vascular system. We will use virus-like particles (VLPs) of wild type (WT) human NoV and
mutants varying in HBGA-binding to investigate binding to lettuce CWM and identify the amino acids involved. The lettuce CWM will be fractionated and tested for NoV VLP-binding to identify the specific plant carbohydrate(s) involved. WT and mutant NoV VLPs will be compared for their persistence on the lettuce leaf to identify whether specific binding enhances entry, transport and persistence. Finally, NoV VLPs and sapovirus (infectious surrogate) will be used to study their entry through roots, trafficking inside lettuce, and interaction with the plant vascular system. Such knowledge will enhance efforts to reduce NoV/SaV contamination in leafy greens, thus will improve public health.
<p>APPROACH:<br/> Since human NoVs are still unculturable in vitro, we will use virus-like particles (VLPs) of human NoVs and surrogate viruses (porcine sapovirus and non-human primate Tulane virus) to perform most experiments. Finally, we will confirm our findings with infectious human NoVs from human diarrhea samples. We will use wild type (WT) human NoV and mutants varying in HBGA-binding to investigate binding to lettuce cell wall materials (CWM) and identify the viral amino acids involved using ELISA assays. An increase or decrease in binding will indicate that the amino acids confer enhanced or reduced attachment to lettuce cell wall carbohydrates, respectively. In collaboration with Dr. Ahmed Fail at Ohio University and the Complex Carbohydrate Research Center, the lettuce CWM will be fractionated into cellulose, hemicelluloses and pectins. We will test individual
fractions for NoV VLP-binding to identify the specific plant carbohydrate(s) involved. Significant differences will indicate that specific carbohydrate fractions contain oligosaccharides capable of binding to NoV VLPs. From this objective, we may identify a naturally occurring plant-derived lectin inhibitor of this binding that could be safe and effective for treating plants to block NoV binding. WT and mutant NoV VLPs will be compared for their persistence on the lettuce leafto identify whether specific binding enhances entry, transport and persistence using real-time reverse-transcription (RT)-PCR. Finally, NoV VLPs and sapovirus (infectious surrogate) will be used to study their entry through roots, trafficking inside lettuce, and interaction with the plant vascular system using light and transmission electron microscopy and cell culture-based infectivity assays. The NoV VLPs and
SaV/Tulane virus particles inside the plant stems and leaves will be observed as electron dense particles with correct size for caliciviruses (27-35 nm in diameter). If a leaf sample is positive in the sapovirus infectivity assay, it indicates that infectious animal enteric calicivirus can be transported from roots to leaves. We have recruited collaborators who are experts in project-related fields to ensure our success of the proposed studies. When we submit original research articles to peer-reviewed journals and presenting our research progress in targeted conferences, we will get peer evaluation and feedbacks to ensure the quality of our research.