Virus Titration via ELISA and EID50 Assays

ELISA (enzyme-linked immunosorbent assay) was used to measure TCID₅₀ titers as previously reported [19 (link)], MDCK cells (ATCC, Manassas, VA, USA) were grown on 96-well flat-bottom microplates at 37 °C with 5% CO₂. MDCK cells with 80–90% confluence were washed with 1× concentrated PBS and then inoculated with serial 10-fold dilutions of virus suspensions in media containing 1 µg/mL of TPCK-trypsin. Virus-infected cells were incubated at 37 °C with 5% CO₂ for 72 h. Next, the TCID₅₀ titers were determined via the Reed and Muench method [20 (link)]. To determine EID₅₀, the allantoic cavities of 10-day-old SPF ECEs were inoculated with 100 μL serial 10-fold dilutions of the viruses, using 5 eggs for each dilution. The eggs were incubated at 37 °C for 96 h. Allantoic fluid was harvested and tested using HA assays [21 ], and EID₅₀ calculation of viruses was completed using the Reed and Muench method [20 (link)].