Figure 1
SeV minigenome expression system. (A) BSR-T7/5 cells were co-transfected with a minigenome plasmid (SVec/HA-M, SVec/HRSV/FTM-, or SVec/HMPV/FTM-) and plasmids encoding for SeV L, N, and P proteins, and infected 24 h later with SeV at a m.o.i. of 5. Cells were harvested 24 h.p.i. and 200 μl of culture supernatant were inoculated into 9-day-old embryonated chicken eggs, which were incubated for 48 h at 34°C, and then transferred to 4°C overnight. Allantoic fluid was harvested and subjected to consecutive passages. (B) The immunofluorescence assay was performed in parallel cultures of BSR-T7/5 cells growing in microchamber slides. Cells were transfected with the indicated minigenome along with support plasmids (upper panels) or with the minigenome plasmid only (lower panels) and fixed and stained 48 hours later. (C) Western blot of allantoic fluid from passages 1–7 (lanes 1–7) are compared with allantoic fluid from uninfected eggs (0). Upper panels were developed with antibodies specific for each of the target proteins (HA-M, HRSV/FTM-, or HMPV/FTM-), middle panels show expression levels of SeV F protein, and lower panels show chicken egg albumin (ova) expression levels, used to standardize the amount of protein loaded in each lane. The asterisk (*) denotes bands that cross-reacted with certain antibodies whose identity has not been investigated. F0 and F1 denote uncleaved F protein precursor and F1 chain, respectively. T7 indicates the bacteriophage T7 promoter and Rz is the hepatitis delta virus ribozyme.