Grapevine Leafroll Associated Virus (GLRaV) types 4 & 6, Molecular Charachterization, Biological Study and RT-PCR Detection

Sequencing of the genomic RNA of Grapevine leafroll associated virus (GLRaV) -4 and -6 was completed. In this effort 13250 and 13811 nucleotides (nt), respectively, for GLRaV-4 and -6 were sequenced. These sequences included all open reading frames (ORF, or functional genes) of the viruses. From the organizational point of view, genomes of these two viruses appeared virtually identical and encoding for 6 viral proteins: replicase-associated protein (encoded by ORF1a and ORF1b by ribosomal frame shift), small hydrophobic protein (p5), HSP-70 homologue (HSP70h), 60K protein (p60), viral coat protein (CP) and protein with a molecular mass of 23K (p23) at the very extreme 3?end. The genome organization of GLRaV-4 and -6 suggests that both viruses belong to the genus Ampelovirus in the Family of Closteroviridae of plant viruses. Amino acid (aa) comparisons of the ORFs showed that the ORF2 (P5) was the most conserved gene among GLRaVs-4, -6 and -9 and showed 89-91%identity. In comparing the aa sequences of the coat protein (CP) of GLRaVs-4, -5, -6 and -9, it showed that the size of the viral coat proteins were slightly different in length and ranged from 268 aa for GLRaV-9 to 272 aa in GLRaV-4. GLRaV-4 and GLRaV-6 CPs are slightly closer to GLRaV-5 and GLRaV-9 (83-85%) than to each other (81%). However, in all cases identity among the viruses was lower than the threshold for the species demarcation established by the International Committee on the Taxonomy of Viruses (ICTV). The phylogenetic analysis also clearly demonstrated that these two viruses clustered with others in the genus Ampelovirus. Furthermore, the data showed that GLRaVs-4, -5, -6 and -9 are clustered together and have closer relationship compared to GLRaV-1 and -3 the other two grapevine viruses in this genus.