Isolation and Characterization of a Novel Gammaherpesvirus from a Microbat Cell Line

Shabman, Reed S.; Shrivastava, Susmita; Tsibane, Tshidi; Attie, Oliver; Jayaprakash, Anitha; Mire, Chad E.; Dilley, Kari E.; Puri, Vinita; Stockwell, Timothy B.; Geisbert, Thomas W.; Sachidanandam, Ravi; Basler, Christopher F.

Transmission electron microscopy. For ultrastructural analysis in ultrathin sections, Myotis bat cells were fixed for at least 1 h in a mixture of 2.5% formaldehyde prepared from paraformaldehyde powder and 0.1% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.3) to which 0.03% picric acid and 0.03% CaCl 2 were added. The monolayers were washed in 0.1 M cacodylate buffer, and cells were scraped off and processed further as a pellet. The pellets were postfixed in 1% OsO 4 in 0.1 M cacodylate buffer (pH 7.3) for 1 h, washed with distilled water, and en bloc stained with 2% aqueous uranyl acetate for 20 min at 60°C. The pellets were dehydrated in ethanol, processed through propylene oxide, and embedded in Poly/Bed 812 (Polysciences, Warrington, PA). Ultrathin sections were cut on a Leica EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL), stained with lead citrate, and examined in a Philips 201 transmission electron microscope at 60 kV.