Figure 1 Generation of recombinant BRSV.

A Diagram of the rBRSV-mCherry cDNA construct cloned in the pACNR1180 low copy vector at NotI site. The synthetic cassette made by DNA synthesis contains the full-length BRSV Lövsta cDNA, a T7 promoter (T7 pr) fused to the Leader region, an HDV ribozyme (RZ) at the 3’ end of the antigenome, followed by a T7 transcription terminator (T7 ter). The expressed antigenomic RNA contains 3 additional guanosines at the 5’ end, an exact 3’ end (Trailer) sequence, and an additional transcription unit to express mCherry in infected cells. B Schematic representation of the protocol of recovery and amplification of rBRSV-mCherry. C Quantification of rBRSV-mCherry replication at 2, 3, and 6 dpi by fluorescence measurement using a Tecan infinite M200Pro spectrofluorometer. BT cells were infected with an undiluted viral stock with a MOI of 0.2 and data are representative of one experiment performed in quadruplicates. Standard deviations are indicated. A representative image of BT cells infected with rBRSV-mCherry at 6 dpi is shown. Cell nuclei were stained with Hoechst 33342. Scale bar, 100 μm. D Quantification of rBRSV-mCherry replication at different MOI from 2 to 8 dpi by fluorescence measurement using a Tecan infinite M200Pro spectrofluorometer. Data were normalized based on the maximal fluorescence intensity obtained and by the value of fluorescence of untreated infected cells. Data are representative of two experiments made in duplicates, except data at 6 dpi which correspond to simplicates. Standard deviations are indicated. E Images of BT cells infected with rBRSV-mCherry (MOI 0.04) at 8 dpi. Cells were fixed and labelled with anti-F antibody (green). Cell nuclei were stained with Hoechst 33342. Scale bar, 100 μm. F, G Comparison of Lövsta and rBRSV-mCherry replication at 3 and 7 dpi. Cells were infected at MOI 0.02, fixed at 3 or 7 dpi and labelled with anti-F antibody, and nuclei stained with Hoechst 33342. Scale bar, 100 μm F Fluorescence was quantified using a Tecan infinite M200Pro spectrofluorometer. Data were normalized by the value of fluorescence of untreated infected cells and are representative of one experiment performed with 6 replicates. Data are means ± SD, ** p < 0.05.