FIG 2.

MVA-NS1 confers protection against multiple serotypes of BTV in IFNAR^−/− mice. (A) Survival rate after infection. Animals were inoculated with 10⁷ PFU of MVA-NS1 or MVA-wt as a negative control following a prime-boost strategy. Afterwards, animals were challenged with a lethal dose of different BTV serotypes. Red circles and continuous line, MVA-NS1-immunized mice; blue circle and dashed line, MVA-wt (B) Viral titers of BTV-1, BTV-4, and BTV-8 recovered in blood of control and immunized IFNAR^−/− mice after challenge. Virus was extracted from blood and determined as described in Materials and Methods. Points indicate group means, and error bars show the SDs. (C) Detection of BTV-4 strain Morocco (BTV-4M) (left) and BTV-16 (right) in blood of control and immunized IFNAR^−/− mice after challenge by RT-qPCR. Total RNA from blood and the expression of mRNA of segment 5 (encoding NS1 protein) were quantified at days 3, 5, and 7 postinfection for BTV-4M and 4, 6, and 10 postinfection for BTV-16. Results are expressed as C_T values and PFU equivalents. Red lines represent the cutoff described by Toussaint et al. (47). (D) Postchallenge sickness scores for control and immunized IFNAR^−/− mice challenged with BTV-4. Animals were evaluated and scored for the following individual signs: rough hair (absent = 0, slight = 1, and marked = 2), activity (normal = 0, slightly reduced = 1, reduced = 2, and severely reduced = 3), eye swelling (absent = 0, slight = 1, moderate = 2, and severe = 3), and temperature (normal = 0 and hypothermia = 3). The final score was the addition of each individual score. The minimum score was 0 for healthy; scores of 1 to11 indicated severity. Animals that reached 10 points were euthanized. Each score represents the value of a single animal. Asterisks indicate statistical significance calculated using the parametric unpaired Student's t test (P < 0.05).