Sequencing and phylogenetic analysis of the complete genome of the Bo/CH/HB/BD/2019 strain
Based on the GenBank sequences, we designed six pairs of specific primers using Primer Premier 5 software. The reference sequences were AY126472.2, JX145650.1, EU794907, AF097917.5, MK159169.1 and NC_029645.1. The complete BNoV genome of the strain Bo/CH/HB/BD/2019 was amplified using six sequence-specific primer pairs.
Reactions were performed in a total volume of 20 μL (2 μL of cDNA, 2 μL of 10× PCR Buffer, 3 μL of 2.5 mM dNTP, 1 μL of each primer, 0.5 μL of Taq polymerase, and 10 μL of ddH2O). The whole genome of BNoV strain Bo/CH/HB/BD/2019 was amplified by RT-PCR (oligonucleotide primers are presented in Table 1). All PCR products were purified with the PCR Purification Miniprep Kit (BIOMIGA, Shanghai, China) following the manufacturer’s instructions. The PCR products were cloned into the pMD19-T cloning vector (TaKaRa Biotechnology, Dalian, China). The positive recombinant plasmids were verified by restriction enzyme digestion and sequenced by Sangon Biotech (Beijing, China). The raw genomic sequence fragments were imported to SeqMan in DNAStar (DNAStar, Inc., Madison, WI, USA) for assembly and annotation. The complete BNoV genome sequence has been deposited in GenBank (accession number: MN480761). Sequencing and phylogenetic analysis of the complete genome of the Bo/CH/HB/BD/2019 strain was performed using the Clustal W program in DNA Star software. Then, 10,000 bootstrap replication was performed using MEGA 7 software, and the phylogenetic tree was constructed by the neighbor-joining method.