More recently, we found that F11-mediated down regulation of RhoA signalling also promotes the release of virus from infected cells in culture. This conclusion was based on the effects of over-expression of dominant negative and active RhoA and mDia clones combined with pharmacological inhibition of RhoA signalling and actin cytoskeleton dynamics. In our current study, we directly examined whether inhibition of RhoA signalling promotes viral release and Lobeline HCl spread using recombinant viruses lacking the F11L gene or expressing an F11 mutant that is deficient in RhoA binding. Our data now clearly demonstrates using an intranasal mouse model of infection that F11-mediated inhibition of RhoA signalling enhances the spread of vaccinia infection not only in cell monolayers but also in vivo. Consistent with previous publications using recombinant viruses with Riociguat (BAY 63-2521) nonsense mutations in the F11L gene, we found that deletion of the majority of the F11L gene and loss of F11 expression did not affect viral morphogenesis. The loss of F11 or its ability to bind RhoA did however result in significantly higher levels of GTP bound RhoA at 8 hours post infection, consistent with the presence of actin stress fibres in DF11L or F11-VK as compared to WR or WR-A36R-YdF infected cells. We also found that the increased RhoA signalling resulted in a significant reduction in the number of actin tails and amount of virus released into the culture medium. This reduction must at least in part be responsible for the smaller plaques formed by the DF11L and F11-VK viruses, as the ability to induce actin tails is know to promote the formation of larger plaques and enhance viral spread. Although plaque size gives a measure of the efficiency of cell-to-cell spread it provides no information concerning the dynamics of the process as it represents a singlefixed timepoint. Given this we imaged viral spread in live cells during plaque formation to assess more directly how F11-mediated inhibition of RhoA signalling enhances the spread of infection.We found that F11 promotes the spread of infection not only by enhancing viral release but by also stimulating the migration of infected cells after their loss of cell-cell adhesion. Our findings support and extend previous suggestions and observations in isolated cells.