Attachment and localization of human norovirus and animal caliciviruses in fresh produce.

Fresh produce is a high risk food for human norovirus (NoV) contamination. To help control this pathogen in fresh produce, a better understanding of the interaction of human NoV and fresh produce needs to be established. In this study the attachment of human NoV and animal caliciviruses (murine norovirus, MNV-1; Tulane virus, TV) to fresh produce was evaluated, using both visualization and viral enumeration techniques. It was found that a human NoV GII.4 strain attached efficiently to the Romaine lettuce leaves and roots and green onion shoots, and that washing with PBS or 200 ppm of chlorine removed less than 0.4 log of viral RNA copies from the tissues. In contrast, TV and MNV-1 bound more efficiently to Romaine lettuce leaves than to the roots, and simple washing removed less than 1 log of viruses from the lettuce leaves and 1-4 log PFU of viruses from roots. Subsequently, the location of virus particles in fresh produce was visualized using a fluorescence-based Quantum Dots (Q-Dots) assay and confocal microscopy. It was found that human NoV virus-like particles (VLPs), TV, and MNV-1 associated with the surface of Romaine lettuce and were found aggregating in and around the stomata. In green onions, human NoV VLPs were found between the cells of the epidermis and cell walls 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. Collectively, these results demonstrate that (i) washing with 200 ppm chlorine is ineffective in removing human NoV from fresh produce; and (ii) different viruses vary in their localization patterns to different varieties of fresh produce.

Effects of Abiotic and Biotic Stresses on the Internalization and Dissemination of Human Norovirus Surrogates in Growing Romaine Lettuce.

Human norovirus (NoV) is the major causative agent of fresh-produce-related outbreaks of gastroenteritis; however, the ecology and persistence of human NoV in produce systems are poorly understood. In this study, the effects of abiotic and biotic stresses on the internalization and dissemination of two human NoV surrogates (murine norovirus 1 [MNV-1] and Tulane virus [TV]) in romaine lettuce were determined. To induce abiotic stress, romaine lettuce was grown under drought and flood conditions that mimic extreme weather events, followed by inoculation of soil with MNV-1 or TV. Independently, lettuce plants were infected with lettuce mosaic virus (LMV) to induce biotic stress, followed by inoculation with TV. Plants were grown for 14 days, and viral titers in harvested tissues were determined by plaque assays. It was found that drought stress significantly decreased the rates of both MNV-1 and TV internalization and dissemination. In contrast, neither flood stress nor biotic stress significantly impacted viral internalization or dissemination. Additionally, the rates of TV internalization and dissemination in soil-grown lettuce were significantly higher than those for MNV-1. Collectively, these results demonstrated that (i) human NoV surrogates can be internalized via roots and disseminated to shoots and leaves of romaine lettuce grown in soil, (ii) abiotic stress (drought) but not biotic stress (LMV infection) affects the rates of viral internalization and dissemination, and (iii) the type of virus affects the efficiency of internalization and dissemination. This study also highlights the need to develop effective measures to eliminate internalized viruses in fresh produce.

Dynamics of moisture diffusivity in solid triacylglycerol matrices.

The effects of lipid formulation in combination with two crystallization conditions, shearing and non-shearing, on the mechanism of water diffusivity were investigated. Five formulations of lipid which varied in fatty acid compositions, solid fat particles, and emulsifier levels were assessed at 5, 20, and 30°C. Polarized light microscopy was used to study microstructural properties of the samples under dynamic and static conditions; and their moisture permeability was examined using gravimetric technique (ASTM E-96). All formulations and crystallization techniques were confirmed to have a large impact on the crystallization properties and dynamics of moisture migration. Dynamic crystallized samples showed altered structures with different patterns of crystalline clusters. Different values of water diffusivity suggested that shearing affected moisture diffusivity of the samples differently. This effect of shearing was more pronounced in the presence of lecithin. A higher migration rate was observed in sheared samples that contained higher amount of long chain saturated fatty acids.

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 pre- and postharvest 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 a human NoV genogroup II genotype 4 (GII.4) strain or 1 × 10(6) to 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 copies were determined by plaque assay and/or real-time reverse transcription (RT)-PCR. For human NoV, high levels of viral-genome RNA (10(5) to 10(6) RNA copies/g) were detected in leaves, shoots, and roots at day 1 postinoculation and remained stable over the 14-day study period. For MNV-1 and TV, relatively low levels of infectious virus particles (10(1) to 10(3) PFU/g) were detected in leaves and shoots at days 1 and 2 postinoculation, but virus reached a peak titer (10(5) to 10(6) PFU/g) at day 3 or 7 postinoculation. In addition, human NoV had a rate of internalization comparable with that of TV as determined by real-time RT-PCR, whereas TV was more efficiently internalized than MNV-1 as determined by plaque assay. Taken together, these results demonstrated that human NoV and animal caliciviruses became internalized via roots and efficiently disseminated to the shoots and leaves of the lettuce.