Attachment, internalization, and dissemination of human norovirus and animal caliciviruses in hydroponically grown Romaine lettuce

Fresh produce is a major vehicle for the transmission of human norovirus (NoV) because it is easily contaminated during both preand post-harvest stages however, the ecology of human NoV in fresh produce is poorly understood. In this study, we determined whether human NoV and its surrogates can be internalized via roots and disseminated to edible portions of the plant. The roots of Romaine lettuce growing in hydroponic feed water were inoculated with 1×10 6 RNA copies/ml of human NoV GII.4 strain or 1-2×10 6 PFU/mL of animal caliciviruses (Tulane virus, TV; and murine norovirus, MNV-1), and plants were allowed to grow for 2 weeks. Leaves, shoots, and roots were homogenized and viral titers and/or RNA were determined by plaque assay and/or real-time RT-PCR. For human NoV, high levels of viral genome RNA (10 5 -10 6 RNA copies/g) were detected in leaves, shoots, and roots at day 1 post-inoculation and remained stable over the 14 day study period. For MNV-1 and TV, relatively low levels of infectious virus particles (10 1 -10 3 PFU/ml) were detected in leaves and shoots at days 1 and 2 post-inoculation, but reached a peak titer (10 5 -10 6 PFU/g) at days 3 or 7 post-inoculation. In addition, human NoV