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

De novo assembly. Following the removal of sequences originating from the Vero host cells, the remaining reads were subjected to analysis by the Velvet assembler using optimized parameters obtained automatically from the multithreaded script VelvetOptimiser (see Methods). The assembly resulted in eight contigs with lengths ranging from 255 to 6834 bases (Table 1 ). In the control sample no relevant contigs were found. Each contig was subjected to a sequence similarity search against the nr/nt nucleotide collection using BLAST. Of these eight contigs, four (contigs 5 to 8) were highly similar to rRNA sequences, while one contig (contig 2) matched the PhiX174 genome. BLAST results for contigs 1, 3 and 4 did not show a complete match with any sequence in the database, with the best hits exhibiting less than 70% identity; contigs 1, 3 and 4 most resembled the L, M and S genomic segments, respectively, of viruses from the Orthobunyavirus genus (Fig. 2) . Notably, the lengths of the contigs were in best agreement with the typical lengths of the Orthobunyavirus genomic segments and substantially differed from those of other genera in the Bunyavirales order (Table 2 ). These results are consistent with the assignment of the generated sequences to the Orthobunyavirus genus, while the low sequence homology to known Orthobunyavirus sequences suggests the classification of this virus as a new species. Notably, the morphology of the virus, as observed by TEM, is consistent with this classification. We named the virus Ness Ziona virus (NZV) after the location it was discovered in. To verify the presence of NZV in the horse plasma, we designed a specific NZV RT-PCR test based on the assembled NZV S genomic segment. The test was conducted on the original plasma before culturing (H234) and after culturing (H234-Vero). A plasma sample taken from the same horse before infection (H210) was used as a control. The presence of the expected 162-bp band confirmed the existence of NZV in the infected H234 plasma and its absence from the preinfection sample ( Supplementary Fig. 3 ).