Complete genome sequence analysis of Grapevine virus B isolated from Inner Mongolia

Abstract:【Objective】The study aimed to obtain the whole genomes of GVB Inner Mongolia isolates and analyze their genome structures and sequence consistency and phylogenetic relationship with the reported GVB isolates.【Methods】The total RNA was extracted from the Shine-Muscat grape leaves by SpectrumTM Plant Total RNA Kit, and the extracted total RNA was reversely transcribed into cDNA using the PrimeScript™ II 1ST Strand cDNA Synthesis Kit. According to the conserved regions of 22 GVB whole genome sequences reported in Genbank database in NCBI, 3 pairs of primers were designed for the amplification of GVB gene fragments. Using cDNA synthesized by reverse transcription as template, the reaction was conducted under conditions of initial 3 min denaturation at 95 ℃, 35 cycles of 95 ℃ for 20 s, the annealing and extension temperatures of the three pairs of primers were 49 ℃ for 20 s, 72 ℃ for 3 min 30 s; 56 ℃ for 20 s, 72 ℃ for 1 min 10 s; 53 ℃ for 20 s, 72 ℃ for 2 min 30 s. The 5’- UTR and 3’-UTR were amplified using the KAPA HiFi HotStart Ready Mix PCR Kit. The the purifica- tion of PCR amplification products was performed following the instructions of agarose gel DNA recovery kit for specific steps. The purified PCR products were ligated and transformed using the zero-background PTOPO-TA cloning kit and E. coli JM109 Competent Cells, and the E. coli JM109 was transformed by heat shock method. The Vector NTI 10.1.1 was used for splicing and correction, and the fulllength genome sequence was obtained. The BLAST algorithm was used to search the NCBI GenBank databases for homologous sequences and ascertain the identity of target gene. DNAMAN was used to analyze the GVB gene sequence. The phylogenetic tree was constructed using the Neighbor- Joining (NJ) method in MEGA 6.0. The RDP4 was used to analyze the recombination of GVB isolates.【Results】The two full-length genomes of GVB Inner Mongolia isolates were successfully obtained by RTPCR combined with RACE technologie, and all sizes were 7609 nt. They were named GVB Hohhot-1 and GVB Hohhot- 2 respectively and submitted to GenbBank with the login number OM103705 and OM103706 respectively. The sequence alignment analysis showed that they all encoded 5 ORFs. The ORF1 encoded a 5133 nt RdRp, ORF2 encoded a 510 nt protein with unknown function, and ORF3 encoded an MP of 972 nt in length, ORF4 encoded a CP with a length of 591 nt, and ORF5 encoded a NABP with a length of 372 nt, 5’non-coding region of 310 nt, 3’non-coding region of 250 nt. Based on the comparison of the nucleotide sequence and the amino acid sequence of GVB isolates, the nucleotide homology of GVB Hohhot- 1 and GVB Hohhot- 2 isolates with 22 reported isolates was 74.9%- 96.5% and 74.8%-96.4%, respectively. On the nucleotide and amino acid levels, the ORF2 had the lowest consistency with the other isolates and the largest variation, and the amino acid sequences of CP gene of 24 isolates had high homology. The phylogenetic tree was constructed from the whole genome sequences of 24 GVB isolates, using Grapevine Virus A (Accession Number: DQ787959) as external reference. The 24 GVB isolates were divided into two groups, the two GVB isolates obtained in this experiment were fused with GVB isolates from The United States (MN716773), Brazil (KX790785, MW357718), China (KF700375), Canada (MZ440726, MZ440728) and Japan (LC617945). The GVB isolates from Canada (MZ440727, MZ440725, MZ440724, MZ440723, KY426923, MZ344593), South Africa (GU733707, EF583906, KX522545, KJ524452), India (MT454065) , Croatia (MF991949), The United States (JX513897) and Italy (NC_003602, X75448) were clustered into another one. On the phylogenetic tree the 24 GVB isolates were divided into four groups. The two GVB isolates obtained in this experiment were fused to the isolates from Canada (MZ440726, MZ440728) and Japan (LC617945). The two isolates from Brazil (KX790785, MW357718) were divided into a group, and the isolates from China (KF700375) were separately divided into a group, and the isolates from Canada (KY426923, MZ440727, MZ440725, MZ440724, MZ440723, MZ344593), South Africa (EF583906, GU733707, KX522545, KJ524452), Croatia (MF991949) , The United States (JX513897, MN716773), India (MT454065), Italy (NC_003602, X75448) were divided into a group. The two isolates obtained from this study were most closely related to the isolate from Canada (accession number: MZ440726), the consistency was 96.5% and 96.4%. No recombination events were detected in the 24 GVB full-length genome sequences by RDP, GENECONV, BOOTSCAN, MACCHI, CHIMAERA, SISCAN, 3SEQ.【Conclusion】The study obtained the complete genome sequences of the two grape virus B Inner Mongolia isolates for the first time. We also analyzed the structure of the virus genome and its sequence consistency and phylogenetic relationship with reported GVB isolates. This study would enrich the genetic database of GVB, and lay a foundation for the further exploration of classification of GVB , the rule of the virus evolution and the subsequent development of infectious cloning in China.