Since the ratio of the protein expression plasmids used at transfection and the time of harvest may have a bearing on the level of VLP formation, a minigenome-based VLP infectivity assay, similar to those described previously [59, 60] was used to determine the relative concentrations of the constituent plasmids, and to determine the kinetics of VLP formation for optimal production. This assay provides only a comparative assessment of VLP formation since it only accounts for VLPs that are able to incorporate and passage minigenomes. However, based on the assumption that the ratio of empty and minigenome-containing VLPs will be equivalent in each reaction, the method provides an indirect means to determine the optimal set of conditions for VLP production as determined by VLP-incorporated minigenomeencoded CAT enzyme activity. Briefly, the steps involved in the VLP infectivity assay were 1) transfection of NiV minigenome construct and co-transfection with full complement of the NiV protein expression plasmids, N, P, L, M, F and G, using Lipofectamine 2000. 2) following replication (48 hours posttransfection), passage of equal volume of VLP-containing transfected cell SUP on to fresh cells previously transfected with N, P and L plasmids and 3), determination of CAT activity in the VLP infected cells 48 hours later. Replication of the VLPincorporated incoming mingenomes based on reporter gene activity indicates the level of particle formation and release, VLP infectivity, and successful minigenome packaging.