Human norovirus (NoV) is responsible for more than 95% of nonbacterial gastroenteritis and over 60% of foodborne illness each year in the US. However, it has been a challenge to conduct research on human NoV because it cannot be grown in cell culture and lacks a small animal model. Despite major efforts, there is no antiviral therapeutic or vaccine for human NoV. In addition, the survival of human NoV is poorly understood. Fresh produce is major high risk food for human NoV-related outbreaks of gastroenteritis because it can easily become contaminated at any point during pre-harvest and post-harvest stages of production. However the ecology, persistence, and interaction of human NoV in produce systems is poorly understood. Determining the mechanisms underlying human NoV fresh produce contamination can facilitate the development of effective preventative and control measures to limit human NoV outbreaks. 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 × 108 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. At day 14 post-inoculation, the titer of MNV-1 and TV in leaves of lettuce plants reached a level of 2.52 ± 0.42 and 4.63 ± 0.69 log10 PFU/g, respectively. Subsequently, the effect of abiotic and biotic stress on viral internalization and dissemination of MNV-1 and TV in Romaine lettuce was 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 to the soil. Plants were grown for 14 days and viral titer in harvested tissues was determined by plaque assay. 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 ×108 PFU/plant. Leaves and berries were harvested over a 14 day period and the viral titer was determined by plaque assay. Over the course of the study, 31.6% of the strawberries contained internalized MNV-1 with an average titer of 0.81 ± 0.33 log10 PFU/g. In comparison, 37.5% of strawberries were positive for infectious TV with an average titer of 1.83 ± 0.22 log10 PFU/g. The level of TV RNA in strawberry tissues was also evaluated using reverse transcriptase quantitative PCR (RT-qPCR). A higher percentage (78.7%) of strawberries were positive for TV RNA with an average titer 3.15 ± 0.51 log10 RNA copies/g. 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. In simple mediums, the receptor binding ability of human NoV was abolished by E-beam and gamma irradiation at dosages of 35.3 and 22.4 kGy, respectively, using the PGM-MB binding assay. As a comparison, TV infectious particles and TV RNA were completely inactivated by E-beam 14.7 kGy and 19.5 kGy, respectively. Infectious particles and TV RNA were completely inactivated by gamma irradiation at dose of 15.4 kGy and 22.4 kGy respectively. Overall, gamma irradiation treatment inactivated human NoV and TV at a lower dosage compared to E-beam treatment. A two-sided E-beam treatment was used to improve the penetration and dose uniformity in whole strawberries inoculated with human NoV or TV. In whole strawberries, TV was completely inactivated by E-beam at 16.3 kGy as determined by plaque assay. No human NoV or TV RNA was detected in strawberry following 28.7 kGy of E-beam treatment using the PGM-MB binding assay. Under the optimized E-beam treatment parameters, viral inactivation in whole strawberries was achieved at comparable dosage levels to simple mediums (PBS). Overall, both human NoV and TV were found to be highly resistant to ionizing radiation.
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). It was found that HPP (400 MPa) inactivation of human NoV and TV was enhanced in simple mediums at a low initial temperature (4°C) and neutral pH (pH 7). A 3.06 log reduction was observed for human NoV RNA when the virus was suspended in PBS at 400 MPa for 2 min at 4°C and pH 7, and infectious TV was completely inactivated at this condition while TV RNA was reduced by approximately 3 logs. Interestingly, both human NoV and TV in strawberry puree were resistant to HPP at 400 MPa for 2 min at the natural pH of the strawberry (pH 4), demonstrating that the food matrix provided significant protective effects on viral inactivation. However, human NoV and TV were completely inactivated in strawberry puree when the pressure level was increased to 600 MPa. Thus, human NoV and TV can be efficiently inactivated by HPP at the commercially acceptable pressure at the optimized processing condition.
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.