Heat Inactivation of Aqueous Viable Norovirus and MS2 Bacteriophage

Human norovirus is a leading cause of acute gastroenteritis often associated with contaminated food or water exposure. Many studies have used morphologically similar viruses, such as MS2 bacteriophage, and molecular detection methods to study the environmental fate and inactivation characteristics, given the historical challenges to culture human norovirus. In this study, we used human intestinal enteroids (HIEs) to analyze the heat inactivation kinetics of viable human norovirus compared to the surrogate MS2 bacteriophage. Norovirus decay rates were 0.22 min-1, 0.68 min-1, and 1.11 min-1 for 50°C, 60°C, and 70°C, respectively, and MS2 bacteriophage decay rates were 0.0065 min-1, 0.045 min-1, and 0.16 min-1 for 50°C, 60°C, and 70°C, respectively. Norovirus and MS2 bacteriophage had significantly different decay rates at all tested temperatures (p = 0.002 – 0.007). No decrease of RNA titers as measured by realtime RT-PCR for both human norovirus and MS2 bacteriophage over time was observed, indicating molecular methods do not accurately depict viable human norovirus after heat inactivation. Overall, our data demonstrate that MS2 bacteriophage is a conservative surrogate to measure heat inactivation and potentially overestimates the infectious risk of norovirus. IMPORTANCE Norovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide. Treatments to inactivate norovirus are critical to reducing the risk associated with contaminated food and water. The recent developments to replicate human norovirus in human intestinal enteroids (HIE) enables the evaluation of heat inactivation kinetics of viable norovirus. Historically, cultivable surrogate viruses, such as bacteriophage MS2, have been used to measure the environmental fate of human norovirus. Our findings indicate that compared to human norovirus, MS2 bacteriophage is a conservative surrogate to measure the effect of heat inactivation. Furthermore, this study corroborates that measuring viral RNA titers, as evaluated by PCR methods, does not correlate with persistence of viable norovirus.