Electron Microscopy Visualization of Virus

Electron microscopy (EM) was performed to observe the virus based on previous studies (Wang et al., 2021 (link)). For this, the allantoic membrane tissue fluid was first centrifuged at 7,000 g for 30 min at 4°C, and then, ultracentrifuged at 110,000 g for 2 h at 4°C (Hitachi Koki Himac CP 100WX, Japan). The pellets were then resuspended in PBS (pH 7.4) and loaded over a preformed 10–60% sucrose gradient, after which the sucrose gradient was centrifuged at 110,000 g for 1 h at 4°C. The purified virus pellets were resuspended in sterile 1× PBS (pH 7.4) buffer and then negatively stained with 2% phosphotungstic acid. The grids were observed under a JEM-100 CX-II electron microscope after blotting and drying (JEOLLTD, Japan).