Molecular Characterization Of10Tobacco Vein Banding Mosaic Virus Isolates From Shandong Province

Tobacco vein banding mosaic virus (TVBMV) is a distinct member of the genus Potyvirus. It mainly infects solanaceous plants and TVBMV may potentially spread quickly and cause serious damages to tobacco production of Shandong proveince. However, research on TVBMV is very limited, with the scarce data thus preventing analysis of evolution of the entire coding regions of TVBMV. Specifically, the HC-Pro, P3,6K1and CP genes have been only moderately studied. Complete genomes of only two isolates, YND and HN39, have been reported. This is negative for us to analyze the genetic structures of TVBMV and develop efficient control.Tobacco leaves with vein banding and yellow mosaic symptoms were extracted from Yishui (YS), Yinan (YN), Juxian (JX), Feixian (FX), Junan (JN), Zhucheng (ZC), Pingyi (PY), Changle (CL), Laiwu (LW) and Yiyuan (YY) of Shandong proveince in the year2009and2010. The complete RNA genome sequences of10tobacco vein banding mosaic virus (TVBMV) isolates from China were determined using five overlapping cDNA clones and5’-RACE. The ten isolates were compared with other isolates available in GenBank to study genetic diversity and population structure of TVBMV, and the results are as follows.1. Nucleotide and amino acid identities of TVBMV showed geographical differentiation among the TVBMV isolates and all the isolates shared much lower identities with Yunnan isolates, while the3’-UTR was more conservative with the highest nucleotide identity (94.05%～100%).2. TVBMV isolates were subjected to phylogenetic analysis with the complete genome sequences, HC-Pro, P3,6K1and CP genes. With more isolate sequences analyzed, the phylogenetic results suggest that TVBMV was divided into geographical origin-specific subgroups. The nucleotide sequences of HC-Pro, P3, and6K1were grouped into two main branches:group I comprised all the isolates except the Yunnan isolates and group II consisted of the latter set. The phylogenetic tree of the CP gene was divided into three groups:most isolates formed group I; the isolates from Japan, United States and Taiwan constituted group Ⅱ; and the Yunnan isolates comprised group Ⅲ. Group I could be divided into three subgroups for HC-Pro, whereas it was divided into two subgroups for P3,6K1and CP.3. Three of the10isolates were found to have undergone recombination and new recombination sites were identified in the TVBMV genome. The recombination sites were at the3’end of CI, the5’-end of NIb, the3’-end of HC-Pro, the5’end of VPg and the central region of6K1. Recombination events between different groups were not found.4. All coding genes were under negative selection and population demography. The dN/dS ratio for all the coding genes being was lower than1.0, suggesting that the ORF of TVBMV was under negative selection. The frequency distributions of the number of pairwise nucleotide differences obtained from the P1～CP sequences were all ragged and multimodal, indicating the long-term stability of the population.5. This study also provides preliminary data on the3’-untranslated region as potentially the best genome sequence for developing transgenic tobacco, since the3’-UTR was more conservative, although TVBMV showed geographical differentiation.