The Effect of Mechanical Damage, Heat and Flooding on Human Norovirus, Tulane Virus and Murine Norovirus Survival on Pre-harvest Lettuce Plants

Introduction: Leafy greens are often implicated in human norovirus (HuNoV) foodborne outbreaks. On the farm, leafy green plants may experience various stressors including mechanical damage, heat and flooding which may affect virus survival.

Purpose: To assess the effect of damage, heat and flooding on HuNoV survival on pre-harvest lettuce plants.

Methods: Murine norovirus (MNV) and Tulane virus (TV) were used as surrogates to assess HuNoV infectivity. Two days prior to viral inoculations, mature lettuce plants were subjected to the following treatments: mechanical damage of outer leaves, heat at 36°C daytime, or flooding with water. All plants, including controls, were then spot inoculated with HuNoV [6 log genomic equivalent (GE)/ml)], MNV and TV [6 log 50% tissue culture infectious dose (TCID₅₀)/ml] and maintained in a growth chamber at 20°C daytime. RNA viral titers were determined after RNase treatment using real-time reverse transcription-PCR. Infectivity titers for TV and MNV were determined using cytopathic effect-based assays. Each treatment was replicated three times per virus type. Titers were determined on post-inoculation days (PID) 1 and 7. Data were analyzed using two-way ANOVA with time and treatment as factors. Statistical significance was defined at P < 0.05.

Results: None of the treatments significantly affected HuNoV RNA titers, MNV or TV infectivity and RNA titers on any date. For all treatments and controls: HuNoV, TV and MNV showed no significant difference in RNA titers on PID7 as compared to PID1, whereas TV and MNV infectivity titers decreased significantly (average 2.87 ± 0.19 TCID₅₀/g). For MNV and TV, time exerted significant effects on infectivity titers accounting for 94.7 and 93% of total variance, respectively.

Significance: Our results suggest that damage, heat and flooding had no effect on HuNoV viral RNA titers on lettuce plants. However, time had a significant negative effect on the survival of infectious MNV and TV.