Identification and genetic characterization of a novel Orthobunyavirus species by a straightforward high-throughput sequencing-based approach

Abstract

Identification and characterization of novel unknown viruses is of great importance. The introduction of high-throughput sequencing (HTS)-based methods has paved the way for genomics-based detection of pathogens without any prior assumptions about the characteristics of the organisms. However, the use of HTS for the characterization of viral pathogens from clinical samples remains limited. Here, we report the identification of a novel Orthobunyavirus species isolated from horse plasma. The identification was based on a straightforward HTS approach. Following enrichment in cell culture, RNA was extracted from the growth medium and rapid library preparation, HTS and primary bioinformatic analyses were performed in less than 12 hours. Taxonomical profiling of the sequencing reads did not reveal sequence similarities to any known virus. Subsequent application of de novo assembly tools to the sequencing reads produced contigs, of which three showed some similarity to the L, M, and S segments of viruses belonging to the Orthobunyavirus genus. Further refinement of these contigs resulted in high-quality, full-length genomic sequences of the three genomic segments (L, M and S) of a novel Orthobunyavirus. Characterization of the genomic sequence, including the prediction of open reading frames and the inspection of consensus genomic termini and phylogenetic analysis, further confirmed that the novel virus is indeed a new species, which we named Ness Ziona virus.

Figure 1

TEM images of viral particles from the supernatant of Vero cells infected with horse plasma. The supernatant was inactivated by Karnovsky solution and negatively stained with 1% phosphotungstic acid. (A) Low magnification (bar represents 1 µm). Viral particles are indicated by arrows. (B) High magnification (bar represents 50 nm). Representative viral particle with a diameter of ~100 nm.

Figure 2

Top BLAST hits of the Velvet-assembled contigs. BLAST analyses were performed using the Velvet-assembled contigs as queries and the nr/nt nucleotide collection as a database. The top ten hits that were identified for contigs 1, 3 and 4 by BLAST are presented as a graphical overview, where for each hit, the locations and lengths of the homologies that were found are shown using a ruler (in bases) based on the query length. The homologies are color coded according to the BLAST scores.

Figure 3

Comparison of the 5′ and 3′ ends of Orthobunyavirus genomic segments. The genomic sequences (L, M or S) of the indicated viruses, which belong to the Orthobunyavirus genus, were aligned by MegAlign Pro software using the MUSCLE algorithm and default parameters. The first 22 bp (5' end) and last 22 bp (3' end) of the alignment are presented using a color-coded background. Accession numbers of each virus are indicated following the name of the virus.

Figure 4

Predicted open reading frames of the Ness Ziona virus (NZV). Open reading frames (ORFs) were predicted using SeqBuilder Pro for the 3 genomic sequences (L, M and S) of the NZV. The potential ORFs (longer than 25 amino acids and containing a start codon) that were identified for each genomic segment are shown. The color code indicates the frame of each ORF, where 1 to 3 and 4 to 6 are the forward and reverse frames, respectively. The ruler (in bases) indicates the positions of the ORFs on the genomic segment. Filled triangles and vertical black lines indicate potential start and stop codons, respectively.

Figure 5

L-segment-based phylogenetic tree of the Ness Ziona virus (NZV) and other Orthobunyavirus members. L-segment sequences of NZV and the indicated viruses were aligned by MegAlign software using the MUSCLE algorithm with default parameters. The resulting phylogenetic tree based on this alignment is presented. NZV is underlined. The calculated distance for each branch is indicated. The accession number of each virus is noted after the name of the virus. Serogroups of the viruses are noted to the right of the figure.