Development of RT-PCR assays for the detection and the resultant phylogenetic analysis of four grapevine vitiviruses based on the coat protein sequences.

Four sets of primers were designed based on the alignment of the complete coat protein (CP) gene sequences of several isolates of four different vitiviruses, i.e. grapevine virus B (GVB), GVD, GVE and GVF, and tested for their efficiency in RT-PCR assays to detect vitiviruses infections in grapevine. The resultant RT-PCR amplicons were sequenced and analyzed for their genetic variability and phylogenetic studies. The results of the RT-PCR assays showed that these primers were highly efficient in detecting different vitivirus isolates in grapevine material originating from ten different Mediterranean countries. In particular, 76 out of 218 tested samples (ca. 35%) were infected with at least one vitivirus. GVE was the most detected (14.7%), followed by GVF (11.5%), GVB (6.9%), and GVD (2.8%). Nucleotide (nt) sequence analysis of the CP genes from this study and Genbank showed that the sequence identity matrixes among isolates of GVB and GVE were the most variable, with nt identity ranging from 77% to 100%, whereas isolates of GVD and GVF showed more conserved nt identities ranging between 82% to 100% and 86.4% to 99.8%, respectively. The phylogenetic trees constructed based on the CP sequences distinguished two main groups of isolates for each vitivirus species, except for the GVD isolates, which did not show any particular subdivision. In general, the distributions of the isolates in the phylogenetic tree were associated with their geographical origin, thus suggesting limited movement of grapevine materials between the different countries. This study reported for the first time: (i) the development of primers based on the complete CP gene sequences for RT-PCR assays for the universal detection of vitivirus species, (ii) the high genetic variability among Mediterranean isolates of vitiviruses and (iii) the presence of GVD in Jordanian vines, of GVE in grapevines from Hungary, Italy, Jordan, Malta and Palestine, and of GVF in grapevines from Afghanistan, Bulgaria, China, France, Hungary, Italy, Jordan, Lebanon and Malta.