2.7 Nucleotide sequencing, sequence and phylogenetic
analysis
In this study, phylogenetic analysis was based in the S1 gene of the
N-DRV strain. The S1 gene was amplified from the cDNA by RT-PCR with
primers N-DRV-F/R, the sequences of the primers were N-DRV-F:
5’-ATGGATCGCAACGAGGTGATAC-3’ (572-593) and N-DRV-R:
5’-CTAGCCCGTGGCGACGGT-3’ (1520-1537), which was designed according to
N-DRV strain N-DRV-XT18. The PCR products were purified by conventional
methods, and then sequenced by BGI Company Ltd (Beijing, China). The
sequence was edited using the EditSeq software of the DNASTAR Lasergene
12 Core Suit (DNASTAR, lnc.) The sequence similarities between the
isolated strains and published sequences using BLAST online searching
program in GenBank (http://blast.ncbi.nlm.nih.gov). We constructed
phylogenetic trees of S1 sequences with MEGA V7.0 program using the
neighbor-joining method and a bootstrap value selected for 1000
replications (Tang and Lu, 2015a).