Genome organization, serology and phylogeny of Grapevine leafroll-associated viruses 4 and 6: taxonomic implications

Complete nucleotide sequences of the type isolate of Grapevine leafroll-associated virus 4 (GLRaV-4) and of an isolate of GLRaV-6 from cv 'Estellat' (GLRaV-6Est) were generated and compared mutually and with related viruses. The genome organization of both viruses resembled that of members of Subgroup I in the genus Ampelovirus (fam. Closteroviridae). The availability of these sequences, along with previously existing data on related GLRaVs, allowed critical review of the taxonomy and nomenclature of these viruses. In phylogenetic analyses, GLRaV-4 and -6Est consistently grouped with GLRaV-5, -9, and -Pr forming a poorly resolved sub-cluster ("GLRaV-4 group") within the genus Ampelovirus. In-depth study showed that genetic distances between these viruses do not exceed the intra-species diversity observed in other closteroviruses. In Western blots, partially purified preparations of GLRaVs -4, -5, -6 and -9 reacted only with homologous monoclonal antibodies, but were all recognized by polyclonal antisera to GLRaV-5 and GLRaV-9. Serological relatedness among these viruses was further confirmed in DAS-ELISA. In immuno-electron microscopy, GLRaV-6 particles appeared uniformly decorated with homologous monoclonal antibodies, whereas GLRaV-2, used as a control, showed "bipolar" morphology of the virion. Results of this study challenge taxonomy and nomenclature of several GLRaVs suggesting that they are divergent isolates of Grapevine leafroll-associated virus 4 and not, as has been assumed, distinct species (definitive and/or putative) in the genus Ampelovirus.