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

Virus isolation and enrichment. Vero cells were infected with the virus-containing plasma and incubated for 6 days until a massive CPE was observed, which was followed by an additional round of viral enrichment. The viral titer in the supernatant was determined by a plaque assay to be 5.7 × 10 7 pfu/ml. TEM revealed the presence of spherical particles that were approximately 100 nm in diameter and had jagged edges (Fig. 1) . These results confirmed the presence of high-titer viruses in the sample. Nevertheless, the morphology observed was not pathognomonic for virus identification and could be associated with several viral families. www.nature.com/scientificreports www.nature.com/scientificreports/ RNA extraction, library preparation and sequencing. The SMARTer Pico RNA Kit we used in this study enables rapid and direct library preparation from very low amounts of starting material. This procedure, with adjustments made in our lab (see Methods), was performed by a single-tube process that involved RNA fragmentation, random priming, first-and second-strand synthesis, and depletion of the ribosomal cDNA originating from the Vero cells. The libraries were sequenced as single reads of 60 nucleotides by a MiSeq instrument, and the sequencing resulted in over 10 million passed-filter reads. The whole process, including RNA extraction, library preparation and sequencing, lasted less than 12 hours. RNA from noninfected Vero cells served as a negative control for the above procedure.