Indirect Immunofluorescence Assay (IFA)

The expression of viral N proteins in PRRSV infected MARC-145 cells were tested by indirect immunofluorescent assay (IFA) as described in our previous study (10). Briefly, the monolayers of MARC-145 cells were infected with the recombinant or parental viruses. At 48 h post-infection (hpi), MARC-145 cells were fixed using cold methanol for 15 min at room temperature and blocked with 3% BSA (fraction V bovine serum albumin) (Roche, Mannheim, Germany) for 30 min. The fixed cells were then incubated with a monoclonal antibody (mAb) against PRRSV N protein (SR30-A) (Rural Technologies Inc., Brookings, SD, USA) for 2 h at 37°C. After washing with PBS, Alexa Fluor 488-conjugated goat anti- mouse IgG or Alexa Fluor 568-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) (1:1000) as the secondary antibody was incubated for 60 min at 37°C. The cells were washed five times with PBS. Finally, images were captured using an Olympus inverted fluorescence microscope fitted with a CCD camera.