Human intestinal enteroids for evaluating the persistence of infectious human norovirus in raw surface freshwater

Globally, human norovirus (HuNoV) is the leading cause of foodborne illnesses. Norovirus transmission to fresh produce can occur via several sources, including contaminated irrigation water. HuNoV RNA has been detected in freshwater resources, but knowledge about virus infectivity is limited due to a historical lack of a HuNoV cell culture. Recently, HuNoV was shown to replicate in human intestinal enteroids (HIE). The objective of this study was to use HIE to evaluate the persistence of infectious HuNoV in raw (i.e. biologically active) surface freshwater. The virus was spiked into freshwater microcosms sampled from three freshwater ponds and then incubated inside an environmental chamber at 20-15 °C and 50-80 % relative humidity (day-night) and 12 h photoperiod. The water was tested for infectious HuNoV, intact HuNoV capsids, indigenous bacteria, and other water quality parameters over a period of 2 weeks. The persistence of infectious HuNoV in the three freshwater microcosms ranged from ≤1 day to ≥7 days. Decay rates for RNA from intact HuNoV capsids ranged from 0.04 to 0.54/day, predicting a 4.2 to 57.5 days, respectively for 1 log reduction. The intact virus showed a significant negative and positive linear relationship with indigenous bacteria and dissolved oxygen, respectively. Using multiple logistic regression, HuNoV RNA >4.4 log genomic equivalent/ml (Cycle threshold values <32) predicted higher probability of detecting infectious HuNoV in contaminated raw freshwater using HIE. Overall, our results provide valuable insights for enhancing quantitative microbial risk assessment models for pre-harvest agricultural water to understand the public health risks associated with the detection of HuNoV RNA in freshwater.