Survival of infectious human norovirus in water and on leafy greens

Human norovirus known commonly as the “stomach flu” is shed in high numbers in feces from infected individuals. Current chlorination treatment of effluents discharged from wastewater treatment plants into surface water may not completely inactivate the virus. Previous research reported the presence of the virus genetic material in surface water. However, it is not known the proportion of infectious virus surviving in surface water and in relation to fecal indicator bacteria such as generic E. coli. It is also not known whether produce irrigated with contaminated water would still harbor infectious norovirus after chlorine washes. Historically, human norovirus was difficult to grow in cell culture and therefore its infectivity could not be determined. A breakthrough occurred in 2016, in which the virus was reported to infect cultured human intestinal enteroids derived from 3D cultures of intestinal stem cells. This cell culture system will be adapted in this proposal to answer critical knowledge gaps regarding the survival of infectious virus in surface water and on lettuce pre and post-harvest.

In the US, the majority of foodborne illnesses (58%) are caused by human norovirus (HuNoV). Lettuce and other leafy greens are most often implicated in the HuNoV outbreaks. Lettuce is the most popular salad vegetable and is almost exclusively used fresh with minimal chlorine washes postharvest. The presence of HuNoV RNA in river water used for irrigation has been demonstrated in numerous studies worldwide. However, in the US, under the current Produce Safety Rule, agricultural water applied to produce is not required to be tested for viruses, thus overlooking a potential threat to the produce industry. The produce rule requires agricultural water to be tested for generic E. coli which may not predict the levels of HuNoVs. Furthermore, it is not evident whether current lettuce chlorine washes is effective against HuNoV. Because HuNoV was difficult to grow in cell culture, limited data exist on the actual infectious viruses in irrigation water, or on pre-and postharvest lettuce washed with chlorine. However, recently a new cell culture system for HuNoV based on human intestinal enteriods (HIE) derived from 3D cultures of intestinal stem cells has been reported. Therefore, the objectives of this proposal are to determine (i) the survival of infectious HuNoV in irrigation water and it’s percent die-off in relation to that of E. coli, (ii) the survival of infectious virus on lettuce under preharvest conditions and (iii) and on post-harvest lettuce following chlorine washes. Irrigation water will be sampled from multiple produce growing regions and used to set microcosms in which HuNoV will be spiked with or without generic E. coli. The microcosms will be incubated under conditions similar to pre-harvest environment and sampled weekly for 2 months. In addition, green house grown lettuce will be spiked with HuNoV and incubated for 2 weeks. Lettuce samples (whole and shredded) spiked with the virus will be subjected to chlorine washes (100 and 200 ppm) for 2 min. Wash water will also be spiked with the virus and chlorinated at 100 and 200 ppm for 2 min to assess whether infectious HuNoV will be inactivated. The infectivity of HuNoV and its RNA titers will be determined using HIE and real-time qPCR as well as the physiochemical and microbiological quality of irrigation and wash water. Knowledge generated from this proposal is expected to benefit the produce industry in better assessing the risks associated with water and food transmission of infectious HuNoV.

The overall goal of this project is to determine the survival of infectious human norovirus (HuNoV) in agricultural water and in relation to indicator organism Escherichia coli as well as its survival on leafy greens under preharvest conditions. Specifically:1. Determine the survival of infectious HuNoV in water and in relation to generic E. coli. 

2. Determine the pre-harvest survival of infectious HuNoV on leafy greens.

This is the first study to show that HIE can be used to recover infectious HuNoV from lettuce and freshwater. Infectious HuNoV persisted for at least 7 days on preharvest lettuce and in freshwater. E. coli did not correlate with HuNoV or HAV in water over a period of 14 days. The naturally occurring freshwater bacteria can negatively influence the survival of human norovirus. Our results highlight the need to identify another fecal indicator that correlates better with human norovirus. Also, more efforts should be placed on preventing water from being contaminated with HuNoV, as the virus may survive long enough in water to contaminate irrigated crops, such as lettuce. Lettuce contaminated in the field with human norovirus poses a risk to consumers as the virus may survive through the duration of this product’s shelf life.