U bottomed plates

In order to titrate the UK661 and F52/70 viruses, 96-well U-bottomed plates (Thermo Fisher Scientific) were seeded with the immortal B cell line, DT40, at a seeding density of 1 × 10⁴ cells per well in 180 μL media. A 10-fold dilution series of the UK661 or F52/70 viruses was added to the cells, with 20 μL of each dilution added to each well in quadruplicate. Cells were incubated at 37°C for 5 days, fixed in 4% paraformaldehyde and stained with a primary mouse monoclonal antibody raised against IBDV VP2 (Wark, 2000 ) and a secondary goat anti-mouse antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific). Wells were marked positive or negative for the presence or absence of virus by immunofluorescence microscopy and the TCID₅₀/mL calculated by the Reed and Muench method (Reed and Muench, 1938 (link)). In order to titrate the PBG98 and chimeric viruses, 96-well plates were seeded with DF-1 cells at a density of 1 × 10⁴ cells per well in 180 μL media. A 10-fold dilution series of the PBG98 or chimeric viruses were added to the cells, with 20 μL of each dilution added to each well in quadruplicate. Cells were incubated at 37°C for 5 days, and wells were marked positive or negative for the presence or absence of cpe and the TCID₅₀/mL calculated by the Reed and Muench method (Reed and Muench, 1938 (link)).