2.3. cDNA Library Construction, Illumina Sequencing, and Sequence Analysis

The dsRNA sample was used for cDNA library construction using the NEBNext^®Ultra^TM RNA Library Prep Kit (Illumina, USA), following the manufacturer’s instructions. The cDNA is end-repaired and adenylated prior to adaptor ligation, library construction, and amplification when using this method. Then, the sequence-ready library was subjected to 8 million of 150 nucleotide (nt) paired-end reads using Illumina Hiseq 4000 technology. The cDNA library construction and deep sequencing analysis were carried out by Shanghai Hanyu Bio-Tech Co., Ltd. In total, 56 contigs were obtained and the N50 length was 1466 nt. The minimum contig length was 500 bp and the minimum coverage was 18. The GC content of the full-length sequence was about 42.37%. The ends and the other parts of the sequences were all confirmed by Sanger sequencing. To obtain the terminal sequences of the virus, rapid amplification of cDNA ends (RACEs) was conducted [21].

Open reading frames (ORFs) were determined using the National Center for Biotechnology Information (NCBI) ORF Finder program (http://www.ncbi.nlm.gov/gorf/gorf.html). The program of BLASTp in the NCBI database was used to search for the conserved domain and similar sequences of the deduced amino acid sequences. Motif searches were performed in three databases, i.e., the PROSITE database (http://www.expasy.ch/), the Pfam database (http://pfam.sanger.ac.uk/), and the CDD database (http://www.ncbi.nlm,nih.gov/Structure/cdd/wrpsb.cgi). Phylogenetic trees were constructed based on the deduced amino acid sequences of the RdRp regions using the maximum-likelihood (ML) method of the Molecular Evolutionary Genetics Analysis (MEGA) software version 6.0 with 1000 bootstrap replicates, as described previously, with minor modifications [26]. Multiple alignments of the sequences of RdRp were performed with the Clustal-X program [27].