Detection and sequence analysis of grapevine virus B isolates from China

The presence of grapevine virus B (GVB) was detected in 188 grapevine samples from China by double antibody sandwich ELISA (DAS-ELISA) and reverse transcription-PCR (RT-PCR). The accuracy of detection by RT-PCR was confirmed by sequencing amplified PCR fragments. Seventeen samples were GVB-positive by DAS-ELISA and five by RT-PCR. The isolate QMW proved to be positive by RT-PCR only, and four isolates (DGWH, DGW, QM, and JFL) could be detected by both methods. Among the five GVB-positive samples detected by RT-PCR, two isolates were originally collected from Henan province and three from Liaoning province. The expected 722 bp DNA fragment, covering partial ORF3 through partial ORF5, was amplified from the five GVB infected samples. Sequence analysis revealed that the molecular variants΄ composition of GVB in the different isolates was complex. Clones of DGWH, DGW, QM, and JFL isolate shared high nucleotide identities, while the identities among the clones of isolate QMW varied. The variants of GVB isolates obtained in this study showed nucleotide identities from 81.1% to 97.9% among themselves, and 79.1% to 98.5% identity with five previously published GVB isolates in NCBI. The alignment of partial ORF3 and the phylogenetic relationships of ORF4 revealed that the molecular variants of Chinese GVB isolates could be clustered into three groups. Only isolate DGW was in the same group with the reported GVB isolates from other countries; the other four GVB isolates in this study were clustered into two groups.