The number of CD3 + CD8 + T cells producing IFN-γ on days 3, 5 and 12 after boosting with rWR-PRRSV-M was determined using flow cytometric analysis. ICS analysis was performed using a FACSCalibur flow cytometer (BD) according to the methods described previously [11] . Twenty mice were tested from the group vaccinated with pVAX1-Ub-M, and five mice from the groups vaccinated with pVAX1 and PBS were tested at each time point. The splenocytes from each vaccination group were counted, pooled, and stimulated in vitro with M protein-derived peptides, as detailed in section 2.12. Ten thousand CD3 + T lymphocytes were acquired per sample and the number of IFN-γ-secretion CD3 + CD8 + T cells were enumerated. The CD3 + CD8 + lymphocytes that expressed IFN-γ following peptide stimulation were considered to be peptide-specific CTLs. Specific CTL responses were evaluated as the increase in the number of CD3 + CD8 + IFN-γ + cells. Reagents used include PerCP-conjugated CD3e, PE-conjugated CD8a and FITC-conjugated IFN-γ, and BD Cytofix/Cytoperm™, all purchased from Becton, Dickinson & Co (BD).