Epon embedding

The fresh tissues were fixed in 2.5% electron microscopy-grade glutaraldehyde, rinsed with 0.2 M sodium cacodylate buffer for one week, and then fixed in 1% osmium tetroxide (Nanjing Zhongjingkeyi Technology, Co., Ltd., Nanjing, China) for 2 h. The tissues were rinsed with 0.2 M sodium cacodylate buffer three times and overnight. Then, the tissues were dehydrated through a graded series (30%, 50%, 70%, and 90%) of ethanol dilutions, 90% acetone, and pure acetone and processed for Epon embedding (Sigma-Aldrich, St. Louis, MO, USA; Merck Millipore, Burlington, MA, USA). Ultrathin sections (60 nm) were stained with uranyl acetate and lead citrate (Nanjing Zhongjingkeyi Technology, Co., Ltd.) and were then observed using an H-7650 electron microscope (HITACHI, Ltd., Tokyo, Japan).

Viroplasm observations was conducted as previously described [43 (link)]. Briefly, infected MA104 cells were washed once with PBS at 12 hpi and collected by centrifugation at 1000× g for 5 min at 4 °C. The precipitated cells were fixed in 2.5% electron microscopy-grade glutaraldehyde, rinsed with 0.2 M sodium cacodylate buffer, and then fixed in 1% osmium tetroxide (Nanjing Zhongjingkeyi Technology, Co., Ltd., Nanjing, China) for 2 h. The cells were rinsed with 0.2 M sodium cacodylate buffer three times and overnight. The cells were then dehydrated through a graded series (30%, 50%, 70% and 90%) of ethanol dilutions, 90% acetone and pure acetone, then processed for Epon™ embedding (Sigma-Aldrich; Merck Millipore). Ultra-thin sections (60 nm) were stained with uranyl acetate and lead citrate (Nanjing Zhongjingkeyi Technology, Co., Ltd.) and were then observed using H-7650 electron microscope (HITACHI, Ltd., Tokyo, Japan).