Genetic characterization of a novel pheasant-origin orthoreovirus using Next-Generation Sequencing

Abstract

A field isolate (Reo/SDWF /Pheasant/17608/20) of avian orthoreovirus (ARV), isolated from a flock of game-pheasants in Weifang, Shandong Province, was genetically characterized being a field variant or novel strain in our recent research studies in conducting whole genome sequencing by using Next-Generation Sequencing (NGS) technique on Illumina MiSeq platform. Among a total of 870,197 35-151-mer sequencing reads, 297,711 reads (34.21%) were identified as ARV sequences. The de novo assembly of the ARV reads resulted in generation of 10 ARV-related contigs with the average sequencing coverage from 1390× to 1977× according to 10 ARV genome segments. The complete genomes of this pheasant-origin ARV (Reo/SDWF /Pheasant/17608/20) were 23,495 bp in length and consist of 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1) encoding 12 viral proteins. Sequence comparison between the SDWF17608 and classic ARV reference strains revealed that 58.1-100% nucleotide (nt) identities and 51.4-100% amino acid (aa) identities were in genome segment coding genes. The 10 RNA segments had conversed termini at 5' (5'-GCUUUU) and 3' (UCAUC-3') side, which were identical to the most published ARV strains. Phylogenetic analysis revealed that this pheasant ARV field variant was closely related with chicken ARV strains in 7 genome segment genes, but it possessed significant sequence divergence in M1, M3 and S2 segments. These findings suggested that this pheasant-origin field variant was a divergent ARV strain and was likely originated from reassortments between different chicken ARV strains.

Fig 1. The illustrations of the homology search results for NGS reads and the sequencing coverage analysis.

A: Total NGS reads homology search result; B: The remapping of NGS raw reads to ARV 10 segments; C: The result of sequencing coverage analysis.

Fig 2. Amino acid alignment of p10 and p17 protein.

A: The p10 protein of Reo/SDWF /Pheasant/17608/20 align with the homologous protein of Reo/PA/Broiler/05682/12 (or PA05682), S1133, 1733, 138 and Reo/PA/Turkey/22342/13 (or PA22342) strains; B: The p17 protein of Reo/SDWF /Pheasant/17608/20 align with the homologous protein of Reo/PA/Broiler/05682/12 (or PA05682), S1133, 1733, 138 and Reo/PA/Turkey/22342/13 (or PA22342) strains.

Fig 3. Phylogenetic trees constructed by avian orthoreovirus (ARV) based on nucleotide sequences of the L-class, M-class and σ-class homologous genome segments or genes.

Note: The Reo/SDWF /Pheasant/17608/20 strain was marked with a red color dot.

Fig 4. The mVISTA method for whole genome nucleotide alignment.

Alignment result of the Reo/SDWF /Pheasant/17608/20 in comparisons with the Reo/PA/Broiler/05682/12 (or PA05682), S1133, 1733, 138, Reo/PA/Turkey/22342/13 (or PA22342) and J18 strains was illustrated; Areas in pink color represent ≥ 90% similarities; and areas in white represent < 90% similarities. The scale bar measures approximate length of the concatenated genome.