Identification and genetic characterization of a novel Orthobunyavirus species by a straightforward high-throughput sequencing-based approach

Shifman, Ohad; Cohen-Gihon, Inbar; Beth-Din, Adi; Zvi, Anat; Laskar, Orly; Paran, Nir; Epstein, Eyal; Stein, Dana; Dorozko, Marina; Wolf, Dana; Yitzhaki, Shmuel; Shapira, Shmuel C.; Melamed, Sharon; Israeli, Ofir

Transmission electron microscopy imaging. For TEM analysis, the supernatant from Vero cells infected with the horse plasma sample was inactivated and fixed using Karnovsky solution (4% paraformaldehyde and 2.5% glutaraldehyde) for 30 min at 25 °C, which was followed by an additional 30-min incubation at 25 °C 39 . Ten microliters of the inactivated supernatant was applied on 300-mesh carbon-coated copper TEM grids treated previously with 1% alcian blue. The samples were incubated for 10 min, and excess liquid was blotted using Whatman paper. The grids were washed three times with distilled water and stained with 1% phosphotungstic acid. After air-drying, the samples were visualized using an FEI Tecnai T12 microscope operated at 120 kV and equipped with a Gatan ES500W Erlangshen camera. Scaling was performed using a standard of known size, which was measured at different magnifications.