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

In this study, we established a rapid and straightforward protocol based on HTS for the identification of unknown RNA viruses from cell culture-enriched clinical samples. Once sufficient viral material is available from culture, our protocol is applicable. The main limitation in HTS-based identification, especially from relevant clinical specimens, such as whole blood, plasma or serum samples, is the high host nucleic acid content, which typically masks the relatively low viral nucleic acid content. Previous protocols 31-34 that utilized HTS to characterize unknown RNA viruses are relatively laborious and time-consuming and are based mostly on tissues containing high viral titers that are obtained postmortem 13, 15, 16 . Cell culture enrichment can overcome this problem to a certain extent; www.nature.com/scientificreports www.nature.com/scientificreports/ however, even after viral enrichment, the host background can be dominant. Cell culture rRNA, which amounts for more than 90% of the total RNA in the culture, significantly contributes to the high background 35 .