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).