We have chosen a minigenome-based functional assay, the VLP infectivity assay (described under Materials and Methods), to determine optimal expression plasmid ratios for efficient VLP formation based on reporter gene readouts. Briefly, 293T cells were transfected with plasmids as shown in Figure 1 . For titrating NiV G, F and M plasmids, increasing concentrations of either G, or F or M expression plasmids were, in turn, co-transfected with fixed concentrations of the other two plasmids. The minigenome and N, P and L plasmids were transfected using a predetermined ratio [57] . The VLP-containing cell SUP was harvested 48 hours post-transfection, clarified by centrifugation, and equal volume from each was passaged onto fresh cell monolayers (VLP-infected cells) previously transfected with the core proteins required to support the incoming packaged minigenomes. The VLP infected cells were harvested 48 hours later and tested for optimal particle production based on incoming minigenome-encoded CAT activity. This time point was chosen because maximal VLP formation was also found to be time dependent and optimal at 48 hours post-passage (data not shown). The reproducibility of the results was verified in an independent repeat experiment. Results presented in Figure 1 show that within the given range, and based on the levels of minigenome-encoded CAT activity, varying the concentrations of G, F and M plasmids had a bearing on VLP formation. CAT activity in the VLP infected cells appeared optimal in the boxed lanes 7 and 8 but further analysis to ensure reporter activity in the linear range (data not shown) indicated that the largest amount of minigenome-containing NiV VLPs were produced when the cells were transfected with the NiV M, F and G plasmid ratios of 3:1:1 as in lane 7. This ratio was used for making all our VLP preparations.