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

The use of HTS for characterization of unknown viral pathogens in relevant clinical samples remains limited, primarily because of the large ratio between the genome sizes of hosts and pathogens. This limitation is especially true of blood-related samples, where white blood cells are abundant. Nonetheless, virus detection in clinical samples by HTS is starting to be increasingly used. Studies that have tried to identify unknown RNA viruses by using HTS have been conducted mostly in less-relevant clinical samples (i.e., brain) and provided diagnoses in shorter time frames because they did not require culture first, while other studies have typically obtained between tens to a few thousands of viral reads, a number that might be sufficient for resequencing against a model organism but not for characterizing novel viruses 12-20 . In this study, an HTS-based approach was applied to identify the origin of a viral pathogen found to be present in plasma obtained from a hyperimmune horse 21 . To this end, we have established a straightforward procedure that includes virus enrichment in cell culture followed by RNA extraction from the growth medium, rapid library preparation, sequencing and in-depth data analyses. By following this procedure, we successfully identified and characterized a novel species belonging to the Orthobunyavirus genus.