To further assess the effect of GA on plaque formation, we used the CMVPT30-GFP strain, which produces cell-free virions. HFF monolayers in 24-well plates pretreated with GA (1–20 µM) for 1 h were inoculated with 50–100 plaque-forming units of CMVPT30-GFP per well.16, (Fig. 1B). The number of plaques were counted after 7 days and the IC50 was determined. As predicted by the previously-determined IC50, at 5 and at 10 µM the antiviral effect was apparent. Furthermore, at 10 µM, only single cells were infected, indicating that there was no cell-to-cell virus transmission. These results may be explained as inhibition of secondary infections or that viral entry may be a target of GA. To verify these results, we compared the inhibition of infection by GA using a plaque reduction assay (PRA) similar to the PRA for ganciclovir (GCV), the preferred antiviral HCMV drug of choice. The results showed that although there was strong inhibition of the virus at 16 µM GCV, there were still visible plaques (>5 adjacent cells showing HCMV cytopathic effect) indicating cell-to-cell transmission of the virions. On the contrary, CMVPT30-GFP infected monolayers that were treated with 10 µM GA only displayed individual infected cells in intact HFF monolayers, (Fig. 1C), thus indicating no cell-cell transmission. We also tested GA C17:1 and GA C13:0. Both GA compounds had a strong inhibitory effect on HCMV infection by PRA, similar to GA C15:1 (Fig. 1D,E). None of the GA structures showed cytotoxicity at their active concentrations, respectively.