Isolation and Characterization of P. chinensis Gill Viruses

Fifteen grams of P. chinensis gill filaments from individuals collected in 2022 were chopped and added with 100 mL of SM buffer (50 mM Tris-HCl, 10 mM MgSO₄, 100 mM NaCl, pH 7.5) with 0.5 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) (Solarbio, Beijing, China). The tissue was homogenized by a homogenizer in an ice bath at a speed of 10,000 rpm for 5 s. These steps were repeated several times (> 3) until a uniform tissue homogenate was obtained. The homogenate was centrifuged at 1400 g for 15 min at 4 °C (CR21GIII, Hitachi, Tokyo, Japan). A 20 mL of SM buffer was added to the pellet and homogenized in an ice bath at 10,000 rpm for 10 s, followed by centrifugation at 6000 g for 15 min at 4 °C. These two supernatants obtained in the above steps were combined, filtered through a 38 μm mesh, and the filtrate was centrifuged at 10,000 g for 25 min at 4 °C. The final supernatant was again filtered through a 38 μm mesh, mixed with an equal volume of SM buffer, and centrifuged at 40,000 g for 2 h at 4 °C (CP100WX; Hitachi, Tokyo, Japan) to pellet viral particles [49 (link)]. Finally, 500 μL of SM buffer was added to the pellet to suspend it.
The supernatant and the pellet suspension were dropped on grids, negatively stained with 2% phosphotungstic acid (pH 6.5, Solarbio, Beijing, China), and observed under a TEM (HT7700, Hitachi, Tokyo, Japan) at 80 kV.