FIG 4.

Protection elicited by ChAdOx1/MVA-NS1-NS2-Nt in IFNAR(-/-) mice against a lethal challenge with BTV-1 and BTV-8. Groups of IFNAR(-/-) mice (n  =  5) were immunized following a heterologous prime-boost regimen consisting of an initial dose of ChAdOx1-NS1-NS2-Nt followed by a second dose of MVA-NS1-NS2-Nt. A group was left untreated (control). Immunized and nonimmunized mice were challenged with a lethal dose of BTV-1 or BTV-8. (A, C) Survival rates after infection. Curves were found statistically significant compared with nonimmunized survival curve as calculated by Log-rank test (P value  <  0.05). (B, D) Detection of BTV-1 and BTV-8 by RT-qPCR in blood of nonimmunized and immunized IFNAR(-/-) mice after viral challenge. Expression of mRNA of segment 5 (encoding NS1 protein) was quantified at 3, 5, 7, 10, and 15 d.p.i. Results were expressed as Ct (left y axis) and PFU/mL equivalents (right y axis and dotted horizontal lines). The real-time RT-qPCR specific for BTV segment 5 was performed as described by Toussaint et al. (83) and mouse blood containing different concentrations of virus were titrated and used as standards (22, 39). Cut-off Ct  ≥  38 (dotted pink line). Points represent individual Ct for each mouse and lines of the corresponding color represent the mean Ct value of each group. Differences between groups were calculated by multiple t test analysis using the Sidak–Bonferroni method. ** P value  <  0.002; *** P value  <  0.001.