Binding of Virus-Like Particles of Norwalk Virus to Romaine Lettuce Veins

Noroviruses (NoV) annually cause millions of cases of gastrointestinal disease in the United States. NoV are associated with raw shellfish outbreaks, particularly oysters, which are thought to bioaccumulate NoV particles during the filter-feeding process. NoV outbreaks, however, have also been known to occur from other common-source food-borne vehicles, such as lettuce, frozen raspberries, and salad. In this study, we evaluated romaine lettuce as a potential vehicle for NoV transmission by testing the binding and distribution of NoV to the surface of romaine. Recombinant Norwalk virus-like particles (rNVLP) applied to the surface of romaine lettuce localized as large clusters primarily on the leaf veins. An extract of romaine lettuce leaves in phosphate-buffered saline (PBS) (romaine extract [RE]) bound rNVLP in a dose-dependent manner. RE did not bind rNVLP by histo-blood group antigens (HBGA), nor was RE competitive with rNVLP binding to porcine gastric mucin. These results suggested that non-HBGA molecules in RE bind rNVLP by a binding site(s) that is different from the defined binding pocket on the virion. Extracts of cilantro, iceberg lettuce, spinach, and celery also bound rNVLP. Samples of each of the vegetables spiked with rNVLP and tested with anti-NVLP antibody revealed by confocal microscopy the presence of rNVLP not only on the veins of cilantro but also throughout the surface of iceberg lettuce.