Competitive Fitness in Coronaviruses Is Not Correlated with Size or Number of Double-Membrane Vesicles under Reduced-Temperature Growth Conditions

Al-Mulla, Hawaa M. N.; Turrell, Lauren; Smith, Nicola M.; Payne, Luke; Baliji, Surendranath; Züst, Roland; Thiel, Volker; Baker, Susan C.; Siddell, Stuart G.; Neuman, Benjamin W.

Electron microscopy. Cells were inoculated with at least 20 PFU of virus per cell and maintained at 33°C. Infected cells were fixed in electron microscopy-grade 4% glutaraldehyde (Sigma) in 0.1 M cacodylate buffer, scraped from the plate, and then postfixed in 0.1 M cacodylate-buffered 1% osmium tetroxide for 1 h. The cells were dehydrated in increasing concentrations of acetone and then embedded in Agar 100 resin (Agar Scientific). Ultrathin sections that produced a silver interference pattern (50 to 60 nm in thickness) were taken from embedded samples and poststained with 0.5% uranyl acetate. Electron microscopy was performed at 80 kV on a Philips CM20 equipped with a 2K charge-coupled devide (CCD) camera or a Hitachi H600 equipped with film. Film negatives were scanned into digital files using a Microtek i800 scanner. Cell sections used here each contained a single visible nucleus, with intact nuclear and plasma membranes. Images of DMV-and virion-containing cells meeting these criteria were captured for later analysis.