Evaluation of Heat Inactivation of Human Norovirus in Freshwater Clams Using Human Intestinal Enteroids

Foodborne disease attributed to the consumption of shellfish contaminated with human norovirus (HuNoV) is one of many global health concerns. Our study aimed to determine the conditions of the heat-inactivation of HuNoV in freshwater clams (Corbicula japonica) using a recently developed HuNoV cultivation system employing stem-cell derived human intestinal enteroids (HIEs). We first measured the internal temperature of the clam tissue in a water bath during boiling at 90 °C and found that approximately 2 min are required for the tissue to reach 90 °C. Next, GII.4 HuNoV was spiked into the center of the clam tissue, followed by boiling at 90 °C for 1, 2, 3, or 4 min. The infectivity of HuNoV in the clam tissue homogenates was evaluated using HIEs. We demonstrated that HuNoV in unboiled clam tissue homogenates replicated in HIEs, whereas infectivity was lost in all boiled samples, indicating that heat treatment at 90 °C for 1 min inactivates HuNoV in freshwater clams in our current HIE culture system. To our knowledge, this is the first study to determine the thermal tolerability of HuNoV in shellfish using HIEs, and our results could be informative for developing strategies to inactivate HuNoV in shellfish.