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

To identify the virus present in the horse plasma, we first employed two programs that specialize in rapid and accurate taxonomic profiling of repertoires of microbial organisms in sequenced samples. Nevertheless, a prerequisite for successful identification of a microbial agent by these tools is the existence of the sequence of the organism in the databases used to analyze the data. In the current study, NZV was discovered to be a novel Orthobunyavirus and thus could not be recognized by these profiling tools. We therefore applied a complementary approach based on de novo assembly that did not require any prior knowledge of the sequences of the organisms present in the sample. We used two different algorithms for the de novo assembly, namely, Velvet and SPAdes, which produced similar results, although Velvet was somewhat more stringent and thus gave slightly shorter contigs. Three of the resulting contigs could be assigned to the L, M and S segments of NZV. After a refinement step, where the contigs were used as references to map the reads, and after filtering out the low-quality alignments, we obtained the complete sequences of the three segments of the virus. Determination of whole-genomic sequences is the gold standard in genomic characterization and allowed in-depth characterization of NZV. The characterization included the prediction of ORFs, identification of the consensus sequences of the genomic termini, and phylogenetic classification; the phylogenetic classification closely resembled the serogroup classification. Notably, segments L and S showed high homology with other Orthobunyavirus members throughout the segments, while segment M exhibited lower homology in the central region and higher homologies in the flanks (Fig. 2) . Segment M of Orthobunyavirus members typically encodes for three polypeptides, Gn, NSm and Gc, which are cotranslationally cleaved. The central region of the M segment encompasses the predicted NSm sequence. The low homology found in the NZV predicted NSm sequence might indicate the uniqueness of the virus and further supports the concept that this is a new member of the Orthobunyavirus genus.