2.6. Thermal Stability Assay

Viral titers from stocks were adjusted to 1.0 × 10⁵ TCID₅₀/_mL, stored at 4 °C or 20 °C for a defined duration for the experiment, and subjected to an infectivity assay that determined TCID₅₀ unit using serial ten-fold dilutions of the sample and HEp-2 cells cultured in a 96-well microplate. After inoculation of each dilutant, the plate was centrifuged at 2000 rpm for 30 min followed by incubation in a 5% CO₂ incubator at 34 °C for 5 days. Subsequently, development of CPE was observed, and the viral titer was calculated according to the Leed and Mench method [17,18].