Virus titration by infectious fluorescent focus assay (FFA)

To determine the infectious titer of virus stocks, PK15-C1 were seeded at 2 × 10⁴/well in 96-well optical-bottom plate (Nunc) and grown overnight at 37 °C to approximately 90% confluency.

The next day, wells populated with PK15-C1 were inoculated with virus stocks from 6 passaged generations serially diluted tenfold in MEM medium supplemented with 2% FBS. Dilutions were prepared in duplicate and inoculum was allowed to infect the cells for 4 h at 37 °C under 5% CO₂. A negative control (non-inoculated cells) was included in all titrations. At 3 days post-infection the confluent monolayers of PK15-C1 cells were fixed with a solution containing 80% acetone and 20% methanol at 4 °C for 20 min. After carefully washing with PBS buffer, the infected cells and controls were incubated with pig anti-PCV2 polyclonal antiserum (VMRD, USA) diluted 1:2000 in PBS buffer supplemented with 1% BSA for 1 h at 37 °C. Cells were then washed 3 times with PBST (0.05% Tween-20 in PBS, pH 7.4), and incubated with fluorescein (FITC)-affiniPure secondary goat anti-swine IgG antibody (Jackson ImmunoResearch) for 45 min at 37 °C in the dark. Finally, cells were washed 3 more times with PBST prior to quantification of the numbers of infected cells (fluorescent foci) under a FITC-fitted fluorescent microscope at 20 × magnification.