Hemolytic Assay

To analyze the activity of the complement system, sheep erythrocytes (1 × 10⁸ cells/ml) resuspended in VBS were sensitized with C5b6 (1 µg) for 60 min at RT using a U-bottom microtiter plate from Greiner Bio-one (Kremsmünster, Austria). In a separate preparation, E protein (10 µg), Vn (20 µg), and mixtures of Vn-and NS1 (containing 20 µg VN and 10 µg NS1) and Vn and-E protein (containing 10 µg E protein and 20 µg Vn) were each incubated with C7-C9 (C7 (1 µg), C8 (0.5 µg), and C9 (1 µg) for 15 min at 37°C. Next, the prepared mixtures were added to the sheep erythrocytes coated with C5b6 and incubated for 30 min at 37°C in a total volume of 100 µl per reaction (9). After centrifugation, the hemolytic activity of the complement system was measured by quantitating the released hemoglobin in the supernatant at 415 nm. To test whether virus particles interfere with complement activation, a two-fold serial dilution of NHS was pre-incubated with ZIKV for 30 min on ice. Sensitized sheep erythrocytes (20 µl, 2 × 10⁸ cells/ml) were added to the samples and the mixture was incubated for 30 min at 37°C. The amount of hemoglobin released from the lysed cells was measured by determining the absorbance of the supernatant at an optical density (OD) of 415 nm. To distinguish between the effects of NS1 or E-Protein on the reduction of hemolysis, viral proteins were incubated separately or as a mixture with NHS (1:160 in VBS) before sensitized sheep erythrocytes were added and the lysis assay was performed as described above.