Viral Titer Determination

Viral titers were determined by measuring the 50% tissue culture infective dose (TCID50) in a microtitration assay using RD cells, as described previously (Yu et al., 2015). Briefly, RD cells were seeded onto 96-well plates and incubated at 37°C for 24 h. Virus-containing supernatant was serially diluted 10-fold and 100 μL were added per well in octuplicate. The cytopathic effects were observed once per day until the experimental endpoint was reached. The TCID50 was determined by the Reed–Muench method (Reed, 1983), which is based on the assumption that 1 × 10⁵ TCID50/mL will produce 0.7 × 10⁵ plaque forming units/mL¹.