Figure 3D showed the changes in viral load at 3, 5, and 7 dpi. Following EV-A71 infection, a rapid and dramatic increase in brain C3 and C5a levels was observed, peaking at 148 hpi (Fig. 3E and F). Fluorescence-activated cell sorting (FACS) analyses confirmed activation of the C5a–C5aR1 axis in astrocytes (Fig. S1). Moreover, we observed that EV-A71 infection increased C1q, C3, C3aR1, C5a, and C5aR1 mRNA levels and the transcription levels of proinflammatory cytokines, including CXCL1, IL-6, IL-1β, TNF-α, and MCP-1, within brain tissue (Fig. 3G). The gene discussed is TNF; the disease is infection.