2.7. Serum Neutralization Test (SNT)

SNTs were performed as previously described [54]. All serum samples were filtered and heat-inactivated for 30 min at 42 °C before testing. First, 200 μL of tilapia serum samples were diluted 4-fold with M199, and then, 2-fold dilution series were prepared with M199 supplemented with 0.15% bovine serum albumin (BSA), resulting in 1/4 to 1/512 dilutions, and 50 μL diluted sera or M199 supplemented with 0.15% BSA was added to 96-well plates (6 wells for one dilution). Next, 50 μL of TiLV-2017A (1 × 10⁵ TCID₅₀/mL) was added to each well of the plate and then incubated at 28 °C for 3 h. After incubation, 100 μL of TiB cell suspension in M199 supplemented with 0.15% BSA (2.0 × 10⁵ to 5.0 × 10⁵ cells) was added to each well of the plates that were then incubated at 28 °C for 7 days. The neutralizing antibody titer of the serum against TiLV was determined as the 50% endpoint of the serum that inhibited CPE in inoculated cells.