Quantitative RT-PCR for Viral Gene Expression

The virus pre-treatment was performed as described above. After 24 h of infection, the total RNA was extracted by the TRIzoL reagent (Thermo Fisher, Waltham, MA, USA) [59 (link)]. Next, 1 µg of RNA was retrotranscribed with a SensiFAST™ cDNA Synthesis Kit (Meridian Bioscience, Washington, DC, USA) to cDNA. Real-Time PCR (RT-PCR) tests were run in triplicate, whereby 2 µL of cDNA was amplified using 2× SensiFAST™ SYBR^® No-ROX Mix (Meridian Bioscience, Washington, DC, USA) and 0.4 µM of primers, at a final volume of 20 µL. The relative target threshold cycle (Ct) values of UL52 and UL27 (for HSV-2), spike (S) protein and RNA-dependent RNA polymerase (RdRp) protein (for HCoV-229E), and spike (S) protein and nucleocapsid (N) protein (SARS-CoV-2) were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. The primers are listed in Table 2. The relative expression levels of the genes of interest were expressed using the 2^−∆∆Ct method.