2.6. Measurement of 50% Tissue Culture Infectious Dose (TCID50) and 50% Egg Infectious Dose (EID50)

The TCID₅₀ (50% tissue culture infectious dose) titers were determined by the enzyme-linked immunosorbent assay (ELISA) as previously reported [31]. Briefly, Madin-Darby Canine Kidney (MDCK) cells were grown on flat-bottom 96-well plates and washed with phosphate-buffered saline and then the cells were inoculated with serial 10-fold dilutions of virus samples. Inoculated cells were incubated at 37 °C, 5% CO₂ and the TCID₅₀ titers were determined for 3 days and calculated by Reed and Muench [32]. To evaluate the growth kinetics of A/Mandarin duck/South Korea/KNU18-12/2018 (H11N9) and A/California/04-005-MA/2009(H1N1) (abbreviation: A/California/04/2009 (H1N1)) virus in vitro, the MDCK cell was infected with the virus at a multiplicity of infection (MOI) of 0.001. After a 1 hour incubation, the virus inoculum was removed, the cells were washed 3 times with PBS and cultured in a media infection medium containing 1 μg/mL l-1-tosylamide-2-phenylethyl chloromethyl ketonetreated trypsin. Supernatants were collected at 12, 24, 36, 48, 60, and 72 hours postinfection (hpi). The virus titer of each supernatant was determined by TCID₅₀ assay in MDCK cells [33].

To measure EID₅₀, serial 10-fold dilutions of the virus were prepared and 100 μL of each dilution was inoculated into the chorio-allantoic cavities of 10-day-old SPF-embryonated chicken eggs. The eggs were incubated at 37 °C for 3 days. Five eggs were infected with each virus dilution. Harvested allantoic fluid was tested for hemagglutination (HA) activity [34] and EID₅₀ [33].