Internalization and Dissemination of Human Norovirus and Animal Caliciviruses in Fresh Produce and Non-thermal Processes to Inactivate Human Norovirus.

摘要

Human norovirus (NoV) is responsible for more than 95% of nonbacterial gastroenteritis and over 60% of foodborne illness each year in the US. The objectives of this research are to determine whether human NoV and its surrogates become internalized via the root of growing produce and disseminated to the edible portion of the plants grown in soil; to determine whether biotic and abiotic stress can affect the rate of internalization and dissemination of human NoV in fresh produce; and to determine whether non-thermal food processing technologies can effectively inactivate internalized viruses while maintaining the fresh quality of produce.;Romaine lettuce grown in soil was inoculated with 2 x 10 8 plaque forming unit (PFU) of human NoV surrogates [Murine norovirus-1 (MNV-1); Tulane virus (TV)] via the roots of plants. Plants were grown for 14 days and leaves, shoots, and roots of each plant were harvested and homogenized and viral titer was determined by plaque assay. It was found that both MNV-1 and TV can efficiently be internalized via plant roots and disseminated to shoots and leaves of lettuce. It was found that drought stress significantly decreased the rate of both MNV-1 and TV internalization and dissemination compared to normal soil conditions. In contrast, flood stress and biotic stress did not significantly impact viral internalization and dissemination. Additionally, the rate of TV internalization and dissemination in soil grown lettuce was significantly higher than that of MNV-1 (p<0.05), demonstrating that the type of virus affects the efficiency of internalization and dissemination.;Next, MNV-1 and TV internalization and dissemination in fruiting plants (strawberry and bell pepper) grown in soil was determined. MNV-1 and TV were inoculated to the soil of growing strawberry plants at a level of 2 x 108 PFU/plant. Leaves and berries were harvested over a 14 day period and the viral titer was determined by plaque assay. The internalization and dissemination of TV in bell pepper plants was also determined as a comparison of two types of fruiting produce. In contrast to strawberries, no or little virus internalization and dissemination was detected when TV was inoculated into bell peppers grown in soil.;Internalized virus in fresh produce would be protected from all surface decontamination strategies. Therefore, there is a need to develop effective intervention strategies to target internalized viral pathogens while maintaining the quality of fresh produce. Two non-thermal food processes, ionizing radiation and high pressure processing (HPP), were evaluated for efficacy against a human NoV GII.4 strain. TV was used as side-by-side comparison because it is cultivable and binds to histo-blood group antigen (HBGA) receptors as human NoV does. Since human NoV cannot be grown in cell culture, the survival of human NoV was estimated by a novel receptor binding assay coupled with RT-qPCR. Human NoV receptor-containing porcine gastric mucin was conjugated to the surface of magnetic bead (PGM-MB), allowing for the discrimination between genomic RNA from virus particles capable of binding to HBGAs while excluding RNA from viral particles unable to bind to HBGAs. It was found that both human NoV and TV were highly resistant to two types of ionizing radiation, electron beam (E-beam) and gamma irradiation.;The effectiveness of HPP in inactivating human NoV and TV was determined using two pressure levels (400 and 600 MPa) at various processing conditions (such as initial temperature and pH). 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) affect the rate of viral internalization and dissemination in Romaine lettuce; (iii) the type of virus affects the efficiency of internalization and dissemination in Romaine lettuce; (iv) virally contaminated soils can lead to the internalization of virus via plant roots and subsequent dissemination to the leaf and fruit portions of growing strawberry plants; (v) the magnitude of viral internalization in fruiting produce is dependent on the type of virus and plant; (vi) human NoV and TV are highly resistant to two types of ionizing radiation technologies, E-beam and gamma irradiation; (vii) HPP is capable of inactivating human NoV GII.4 and TV at commercially acceptable pressure conditions and is a promising technology to inactivate the internalized virus particles. (Abstract shortened by UMI.).