Ball Python Nidovirus: a Candidate Etiologic Agent for Severe Respiratory Disease in Python regius

Stenglein, Mark D.; Jacobson, Elliott R.; Wozniak, Edward J.; Wellehan, James F. X.; Kincaid, Anne; Gordon, Marcus; Porter, Brian F.; Baumgartner, Wes; Stahl, Scott; Kelley, Karen; Towner, Jonathan S.; DeRisi, Joseph L.

Transmission electron microscopy. Formalin-fixed lung samples from BP1 and BP6 were cut into 1-mm cubes and transferred to 4% paraformaldehyde-2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.24. Fresh lung from BP6 was also cut into small cubes and fixed directly in 4% paraformaldehyde-2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.24. Fixed tissues were processed with the aid of a Pelco BioWave laboratory microwave (Ted Pella, Redding, CA, USA). The samples were washed in 0.1 M sodium cacodylate buffer, pH 7.24, postfixed with buffered 2% OsO 4 , water washed, and dehydrated in a graded ethanol series, 25%, 50%, 75%, 95%, and 100%, followed by 100% acetone. Dehydrated samples were infiltrated in graded acetone-Spurrs epoxy resin (Electron Microscopy Sciences, Hatfield, PA) 30%, 50%, 70%, and 100% and cured at 60°C. Cured resin blocks were trimmed, thin sectioned (70 nm), and collected on Formvar-coated copper grids, poststained with 2% aqueous uranyl acetate and Reynolds lead citrate. Sections were examined with a Hitachi H-7000 TEM (Hitachi High Technologies America, Inc., Schaumburg, IL), and digital images were acquired using a Veleta 2k by 2k camera and iTEM software (Olympus Soft-Imaging Solutions Corp., Lakewood, CO).