Fresh produce is a high risk food for human norovirus (NoV) contamination, because it can easily become contaminated at both the pre- and post-harvest stages of cultivation. Disease surveillance has shown that human NoV is attributed to 40% of all fresh produce related outbreaks reported each year in the U.S. However, the ecology, persistence, and interaction of human NoV and fresh produce are all poorly understood. Increasing outbreaks of viruses in fresh and fresh-cut vegetables and fruits give high urgency to understanding the interaction of human NoV with fresh produce in order to develop effective preventive measures. In this research, the attachment, uptake, internalization, and dissemination of human NoV and its surrogates (murine norovirus, MNV-1; and Tulane virus, TV) were evaluated.
First, the attachment of human NoV surrogates to fresh produce was visualized using confocal microscopy. Purified human NoV virus-like particles (VLPs), TV, and MNV-1 were conjugated with biotin, and subsequently applied to either Romaine lettuce or green onion. The biotinylated virus particles were visualized by incubation with streptavidin coated Quantum Dots (Q-Dots 655), which emit fluorescence that can be viewed using a confocal microscope. It was found that all three surrogates attached to the surface of Romaine lettuce leaves and were found aggregating in and around the stomata. Similarly, human NoV VLPs, TV, and MNV-1 were found to attach to the surface of Romaine lettuce roots. In the case of green onions, human NoV VLPs were found between the cells of the epidermis of both the shoots and roots. However, TV and MNV-1 were found to be covering the surface of the epidermal cells in both the shoots and roots of green onions. The results indicate that different viruses vary in their attachment patterns to different varieties of fresh produce.
A quantitative assessment of the level of attachment of a human NoV GII.4 strain, TV, and MNV-1 was executed using Romaine lettuce as a model system. Romaine lettuce roots and shoots were inoculated with varying levels of TV and MNV-1 and then washed with PBS to remove unattached viruses. It was found that simple washing removed less than 1 log of viruses from the shoots and 1-4 log of viruses from the roots, demonstrating that TV and MNV-1 bound more efficiently to Romaine lettuce leaves than to the roots. A human NoV GII.4 strain was inoculated at a level of 7 log RNA copies/g to Romaine lettuce leaves and roots, and then washed with either PBS or 200ppm of chlorine. The human NoV GII.4 strain was found to attach similarly the both the Romaine lettuce leaves and roots, and that washing with 200ppm of chlorine removed less than 1 log of viral RNA copies from the tissues. The results demonstrate that different viruses attach differently to Romaine lettuce, and that washing is ineffective in removing viral contamination from fresh produce.
Next, it was determined whether human NoV and its surrogates could 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 6 log RNA copies/ml of human NoV GII.4 strain or 6 log PFU/mL of human NoV surrogates(TV and MNV-1), and plants were allowed to grow for 2 weeks. Leaves, shoots, and roots were harvested at days 0, 1, 3, 7, and 14 after virus inoculation. The plant tissues 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 (5-6 log 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 (1-3 log PFU/ml) were detected in leaves and shoots at days 1 and 2 post-inoculation, but reached a peak titer (5-6 log PFU/g) at days 3 or 7 post-inoculation. In addition, human NoV had a rate of internalization comparable with TV as determined by real-time RT-PCR, whereas, TV was more efficiently internalized than MNV-1 as determined by plaque assay. To further confirm the viral internalization via lettuce roots, an identical experiment was performed with the exception that the harvested plant tissues were submerged in 50 ml of 1000 ppm chlorine for 5 min to eliminate any possible viral contaminations. The results showed that there were no significant differences observed in viral internalization in chlorine treated shoots and leaves on any of the study days compared to the untreated samples (P>0.05) during the experimental period. Taken together, these results demonstrated that human NoV and animal caliciviruses attached tightly to roots, became internalized via roots, and efficiently disseminated to the shoots and leaves of the lettuce.
In summary, this research elucidates a major gap in our understanding of the ecology of human NoV in fresh produce, specifically, our understanding of the fate of human NoV after attaching to roots of growing lettuce. Elucidation of the mechanism of virus-plant interaction will facilitate the development of novel interventions to prevent viral attachment and internalization in plants.