FIG. 4.

Cytotoxicity of purified VP5. (A) Cell cytotoxicity was determined at pH 5.5 and 7.5 of L15 media using 5 μg of purified VP5. As controls, two other purified BTV proteins, VP2 and VP7, and particles, virions, and CLPs were used in parallel. In addition, VP5 denatured by heating at 65°C for 30 min (dnVP5) was used as a negative control. The percent cytotoxicity was measured by the amount of LDH release per 10⁴ L929 cells or per 2 × 10⁴ C6/36 cells induced by each preparation as described in Materials and Methods. (B) Effect of increasing amounts of VP5 added onto L929 cells and C6/36 cells at pH 7.5. Readings from quadruple wells of each preparation were used to calculate the percent cytotoxicity. The percent cytotoxicity was calculated by the substitution of the mean absorbance values at 492 nm divided by the maximum release of LDH as described in Materials and Methods.