To further confirm that the NSs exerted an efficient viral replication in the cells that underwent ActD-induced host transcriptional suppression, 293 cells, which showed higher RNA transfection efficiencies than did VeroE6 cells (data not shown), were infected with rMP12-rLuc at an moi of 2. After virus adsorption, infected cells were mock-transfected or independently transfected with in vitro-synthesized RNA transcripts encoding chloramphenicol acetyltransferase (CAT), MP-12 NSs, or wt RVFV ZH501 NSs. Then, the cells were mock-treated or treated with ActD. Analysis of rLuc activities at 16 h.p.i. demonstrated that NSs expression had little effect on rLuc activities in the absence of ActD (Figure 2A ). In the presence of ActD, rLuc activities were clearly higher in the cells transfected with MP-12 NSs RNA transcripts or ZH501 NSs RNA transcripts than in those transfected with CAT RNA transcripts or in the mocktransfected samples (Figure 2A ). In NSs RNA-transfected cells, similar levels of rMP-12-rLuc titers were observed in both the ActD-treated and mock-treated samples, whereas the rMP12-rLuc virus titers in mock-transfected cells and CAT RNA-transfected cells were significantly lower in the presence of ActD compared to the ActD-untreated cells ( Figure 2B ). Western blot analysis showed that NSs proteins were indeed expressed in the cells transfected with RNA transcripts encoding MP-12 NSs or wt RVFV ZH501 NSs ( Figure 2C , lanes 3, 4, 7, and 8). Also NSs protein expression increased the accumulation of rMP12-rLuc N protein in the presence of ActD ( Figure 2C, lanes 7 and 8) . These data demonstrated that NSs exerted efficient rMP12-rLuc replication in the presence of ActD. We noted that ActD treatment modestly reduced the accumulation of the CAT protein ( Figure 2C, lane 6) . Probably large amounts of CAT RNA transcripts that were introduced into the cells partly overcame the translational suppressive effects that were induced by the combination of rMP-12-rLuc replication and ActD treatment.