Long-lasting protection of immunized IFNAR(-/-) mice against a lethal challenge with BTV-4M. Groups of IFNAR(-/-) mice (n = 5) were immunized with (a,b) a single dose of 108 IU per mouse of ChAdOx1-NS1 or ChAdOx1-NS1-Nt or (c,d) following a heterologous prime-boost regimen consisting of an initial dose of 108 IU per mouse of ChAdOx1-NS1 or ChAdOx1-NS1-Nt (prime) followed by a second dose of 107 PFU per mouse of MVA-NS1 or MVA-NS1-Nt (boost), respectively, administered 28 days apart. Immunized and non-immunized mice were challenged with a lethal dose (10 PFU) of BTV-4M at day 160 post-immunization. (a,c) Survival rates after infection. Statistical differences between the curves were calculated using Log-rank test. (b,d) Detection of BTV-4M in blood of non-immunized and immunized IFNAR(-/-) mice after challenge by RT-qPCR. Expression of mRNA of segment 5 (encoding NS1 protein) was quantified at 3, 5, 7, 10 and 17 d.p.i. Results are expressed as Ct (left y-axis) and PFU equivalents (right y-axis and dotted horizontal lines). Cut-off Ct ≥ 38 (dotted pink line). Differences between groups were calculated by multiple t test analysis using the Sidak–Bonferroni method. ** p-value < 0.02; *** p-value < 0.001.