L-RdRp gene codon optimization and recovery of CCHFV from DNA.

<p>(A) Reporter minigenome luciferase activity was measured 48 h after transfecting BSR-T7/5 cells seeded in 10 cm² wells. Cells were transfected with 250 ng of pC-L or pC-L opti, together with 500 ng of pC-N, 50 ng of pT7-M-Renilla [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004879#ppat.1004879.ref019" target="_blank">19</a>], and 30 ng of internal control pGL3 per well. Data are represented as fold increase in <i>Renilla</i> luciferase expression over control transfections in which pC-L was omitted. Error bars indicate means ± standard deviation (n = 3) (B) V5-tagged L-RdRp and N protein levels in cell lysates from panel A as described before [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004879#ppat.1004879.ref009" target="_blank">9</a>]. (C) BSR-T7/5 cells were transfected as presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004879#ppat.1004879.s001" target="_blank">S1C Fig</a> (pC-L support, upper panel), and also with pC-L opti in place of WT pC-L (lower panel). Four days post transfection, BSR-T7/5 cell supernatants were passaged onto SW13 cells, and viral antigens were detected with a GP38 domain-specific mAb (BSR-T7/5 IFA). Three days after passaging, viral cytopathic effect and CCHFV were visualized by bright field (SW13) or immunofluorescence (SW13 IFA) microscopy with anti-CCHFV hyperimmune mouse ascetic fluid (HMAF).