Peripheral Blood Lymphocyte Proliferation and Cytokine Analysis

The peripheral blood lymphocyte proliferation assay was performed using a modified CCK-8 method as described previously^{29 (link)}. Briefly, peripheral blood T lymphocytes were isolated using a Peripheral Blood Lymphocyte Separation Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. After cell counting, 80 μL of diluted cells in RPMI 1640 medium (Gibco) was seeded into a 96-well plate. To specifically stimulate the proliferation of T lymphocytes, 20 µL of CHN-YC virus (10^6.25 TCID₅₀/100 µL) or purified recombinant truncated E protein (20 mg/mL) was added; the unstimulated control was administered an equal volume of PBS. After 36 h of cell culture at 37 °C, cell proliferation was detected using a Cell Counting Kit-8 (MCE, Shanghai, China) according to the manufacturer’s instructions. The stimulation index calculated from the OD value of each stimulated group, and the OD of mock group is normalized to 1.
The levels of Th1-type cytokine IFN-γ and the Th2-type cytokine IL-4 in serum were measured using duck IFN-γ and IL-4 sandwich ELISA kits (ML Bio, Shanghai, China) following the manufacturer’s instructions.