Porcine Mx1 expression alters influenzavirus NP synthesis (A–B) and production of infectious progeny particles (C). A–B: flow cytometric determination of NP-positive counts in Vero cells transfected with plasmids (pcDNA4) encoding either eGFP (negative control), porcine Mx1 (poMx1α) or human MxA (huMxA, positive control). (A) Example. Cell counts (vertical axis) refer to infected (NP-positive) and noninfected (NP-negative) cells detected in induced (transgene-expressing, dark grey) and non-induced (nonexpressing, light grey) cell populations. (B) Percent NP-positive cells 5 hpi in transgene-expressing (black boxes) and nonexpressing (white) cells. The corresponding plasmids had been transfected 24 h before infection, each population process resulting in a mixed population of expressing and nonexpressing cells. Cells were permeabilized, double immunostained for Mx proteins and virus NP and analysed using the FACS Canto, gating on the forward and side scatter to exclude debris and collecting fluorescences in FL-1 and FL-5. A minimum of 10 000 events were acquired and analyzed with BDFACSDiva software v4.1.1. Each box pair (white/black) represents means ± SD in nonexpressing/expressing cells from 3 independent experiments. Expression of both porcine and human MxA resulted in a significant decrease of NP-positive cell counts at the timepoint studied (5 hpi). (C) Influenza virus yields from stably-transduced porcine Mx1-expressing Vero cell monolayers were significantly decreased after induction of poMx1 expression. Pools of induced (black boxes) and non-induced (white boxes) V50 cells were infected with the H1N1 virus for 48 h (m.o.i. = 0.01). The viral titers in the culture supernatants are plotted, as determined in triplicate by standard median tissue culture infectious dose assays. TCID50: 50% tissue culture infective dose. Plotted values are means ± SD of 3 independent experiments. *Significantly different from the titer retrieved from the corresponding non-induced cells, p < 0.05. (A color version of this figure is available on line at www.vetres.org.)