Partial characterization of two divergent variants of Grapevine leafroll-associated virus 4

Two non mechanically transmissible viruses with filamentous closterovirus-like particles were extracted and partially characterized from leafroll-affected grapevine accessions of cv Koussan from Turkey (Y253) and cv Koudsi from Israel (Y252), both from the reference collection of grapevine viruses and virus-like diseases at the Institut National de la Recherche Agronomique (INRA) in Colmar, France. These viruses, denoted Y253-TK and Y252-IL, were independently investigated in France (INRA-Colmar) and Italy (DPPMA). Y253-TK did not react in DAS-ELISA or RT-PCR assays that would have detected any of the Grapevine leafroll-associated viruses (GLRaVs) described so far; Y252-IL did give weak and inconsistent positive reactions in IEM only with an antiserum to strain LR106 of Grapevine leafroll-associated virus 4 (GLRaV-4). A polyclonal antiserum to isolate Y252-IL decorated the homologous particles and, those of Y253-TK and, at lower dilution, particles of isolate GLRaV-4 LR106. A segment of the heat shock protein p70 homologue (HSP70h) gene, was amplified by RT-PCR of RNA from denatured purified particles of Y253-TK by using a set of degenerate primers. The 183 amino acid polypeptide deduced from the 549 bp HSP70h gene fragment showed a high degree of identity with the cognate genes of GLRaV-4 (95%), GLRaV-5 (91%), GLRaV-6 (84%), and GLRaV-9 (90%), but low identity with those of other GLRaVs (33-35%). ne HSP70h of isolates Y252-IL and LR106 showed 95% amino acid identity. The coat protein sequence of isolates Y253-TK showed 99 and 94% amino acid identity with those of isolates Y252-IL and LR106, respectively, with changes concentrated in the N-terminus, a feature that might have a bearing on the reaction with their heterologous antisera. Based on serological and molecular data, it was concluded that Y253-TK and Y252-IL are distinct, though similar, isolates of GLRaV-4. In two independent surveys in which 110 and 320 grapevine accessions from germplasm collections at Colmar and Bari, respectively, were examined by DAS-ELISA, the antisera As-Y253-TK and As-Y252-IL cross-reacted with the homologous and hererologous viruses. Whereas As-Y253-TK did not react with any of the other accessions from the French collection tested, As-Y252-IL recognized a virus present in a total of eight grapevine accessions from the Mediterranean area, grown in the Italian germplasm collection.