PRRSV Virus Titration Protocol

IPMA was used to assess the virus titration as previously described^{48 (link)}. Briefly, cells were fixed with 100 μl of 4% cold formalin for 15 min at room temperature (RT). The fixed cells were washed with 100 μl phosphate-buffered saline (PBS) once after that and twice with 100 μl of 0.5% PBS Tween-20 (PBST) and then blocked with 100 μL of 1% bovine serum albumin (BSA) in 0.5% PBST for 30 min at RT. Then, the cells were washed. Seventy microliters of anti-PRRSV NC protein monoclonal antibody (Median Diagnostics, Gangwondo, Korea) diluted at a ratio of 1:400 was used to stain cells at RT for 60 min. The cells were washed and incubated again with 50 μl of peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson Immuno Research, Pennsylvania, USA) at a dilution of 1:600 for 60 min at RT, washed and counterstained with 1,5-diaminopentane (DAP) substrate for 5 min, washed with distilled water and examined under a microscope. Virus titer was determined using the Reed–Muench method^{46 (link)}, also expressed as TCID₅₀, described as diluting a virus required to infect 50% of a given cell culture^{49 (link)}.