An RTCA-based assay as an innovative approach for thermal inactivation studies of hepatitis A virus

Hepatitis A virus (HAV) is responsible for acute viral hepatitis worldwide, mainly transmitted via the fecal-oral route either through direct contact or by consuming contaminated food. Thermal inactivation is widely applied in the food industry to inactivate viruses and ensure the safety of food products. The effectiveness of inactivation treatments depends on the initial viral load and the sensitivity of the method quantifying infectious viruses. This study aimed to assess the real-time cell analysis (RTCA) assay in the framework of heat inactivation studies, compare its suitability to the traditional PFU assay and molecular-based methods (RT-qPCR, integrity-RT-qPCR), and model thermal inactivation kinetics. Our results showed that the RTCA assay is a suitable method for quantifying the decrease in infectious HAV as the time-temperature scale increases. No more infectious virus was detected after treatment at 37°C, 50°C, 65°C, 72°C, and 80°C, lasting 9 days, 24 h, 10 min, 5 min, and 2 min, respectively. The Geeraerd model was identified as the best fit to describe HAV thermal inactivation kinetics. The D values, corresponding to the time required to achieve a 1 log₁₀ reduction in HAV titer, were comparable between both cell-culture-based methods across all tested temperatures. In contrast, molecular methods yielded significantly higher D values. Kinetic parameters were subsequently validated, confirming that the isothermal-derived parameters can reliably predict viral inactivation under dynamic thermal conditions. This study demonstrates that the RTCA assay provides a powerful tool for modeling HAV thermal inactivation that can offer valuable guidance for implementing effective food safety strategies.IMPORTANCEThere has been a significant increase in viral foodborne outbreaks following the consumption of raw or minimally processed foods. Thermal treatments may ensure food safety, with efficacy depending on viral load and method sensitivity. In this study, we present a powerful real-time cell analysis assay that advances our analytical ability to quantify infectious hepatitis A virus. By providing kinetic parameters comparable to those obtained with the traditional infectious titration method, this high-throughput assay overcomes its limitations to conduct viral inactivation studies on a larger scale. In addition, the successful validation of kinetic parameters confirms that isothermal-derived parameters can reliably predict viral inactivation under dynamic thermal conditions. This offers a reliable and essential tool to strengthen food safety measures to be applied in the food industry.