Rupestris stem pitting associated virus isolates are composed by mixtures of genomic variants which share a highly conserved coat protein

Abstract

Broad spectrum primers were used to amplify a fragment comprising the CP gene and putative ORF6 by RT-PCR from ds-RNA templates originating from 46 Portuguese varieties, totalling 190 samples, including some wild Vitis ssp sylvestris vines, and 2 vines from Slovenia. SSCP analysis was used as a preliminary screen to avoid cloning and sequencing very similar variants. Four groups of variants were recognized. In pair wise comparisons between nucleotide sequences the minimal homology found was 81%. In case of the cultivated varieties, no relationship could be seen between the phylogenetic groups and geographic origin or grape variety. Several isolates were found harbouring mixed infections with genomic variants from different groups, but the mixing did not lead to an extensive recombination between them. The deduced amino-acid sequences revealed a conserved CP subjected to strong purifying selection pressure. Analysis of the selection pressure operating on the putative ORF6 suggests that this ORF does not exist. Previously produced polyclonal antiserum raised against the recombinant CP of RSPaV expressed in Escherichia coli was shown to be able to detect all four groups of variants of RSPaV included in this study, which might enable the diagnosis of the virus on a serological basis.

Sequence diversity, population genetics and potential recombination events in grapevine rupestris stem pitting-associated virus in Pacific North-West vineyards

“…A phylogenomic approach was used to analyse a total of 159 full-length CP sequences (84 from this study and 75 from GenBank) and 94 sequences specific to the HR of RNA-dependent RNA polymerase (RdRp) (57 from this study and 37 from GenBank). As only a few isolates from the USA and none from the PNW were included in previous studies, the work represents an analysis of GRSPaV isolates at a global level, extending previous investigations conducted in other grape-growing regions of the world (Meng et al, 2006;Nolasco et al, 2006;Habili et al, 2006;Lima et al, 2006;Nakaune et al, 2008). These comparative results show that the genetic diversity of GRSPaV in the PNW vineyards is considerably greater than reported in other regions, probably as a result of the introduction of planting materials from several sources outside the region.…”

“…We designated each of these lineages with a reference isolate to maintain a standardized nomenclature of GRSPaV sequence variant groups in analogy with a previous report by Meng et al (2006). Thus, GRSPaV-1, GRSPaV-SG1, GRSPaV-BS and GRSPaV-VS lineages correspond to groups 2b, 2a, 3 and 1, respectively, as proposed by Nolasco et al (2006) and Nakaune et al (2008). The GRSPaV-1, GRSPaV-SG1, GRSPaV-BS and GRSPaV-VS lineages contained 11, 12, 21 and 22 sequences from PNW, respectively, and 20, 10, 15 and 29 from GenBank, respectively.…”

“…The aetiological relationship of GRSPaV with RSP, however, remains to be elucidated. Previous studies have shown that GRSPaV can occur as distinct variants (Rowhani et al, 2000b;Habili et al, 2006;Meng et al, 2006;Nolasco et al, 2006;Nakaune et al, 2008), some of which may not elicit RSP symptoms when graft-inoculated on the indicator 'St George' (Meng et al, 1999(Meng et al, , 2006Habili et al, 2006). In addition, some variants that are latent in Vitis vinifera cultivars and in most American Vitis species and hybrids can produce necrosis of the veinlets when graft-inoculated on V. rupestris 6 Vitis berlandieri 110 Richter (Bouyahia et al, 2005).…”

Variabilidade do gene da proteína capsidial de três espécies virais que infectam videiras no Brasil

“…Através da análise da identidade de nt desses isolados de RSPaV, pôde-se verificar a formação de quatro agrupamentos distintos, à semelhança de outros trabalhos (Lima et al 2006;Meng et al, 2006;Nolasco et al, 2006;Nakaune et al 2008 1 -Árvores filogenéticas não enraizadas, obtidas com o programa MEGA 4.1, baseadas nas sequências completas de nucleotídeos do gene da proteína capsidial dos isolados de (A) Rupestris stem pitting associated virus, RSPaV, (B) Grapevine leafroll associated virus 2, GLRaV-2 e (C) Grapevine fanleaf virus, GFLV, com a demarcação de distintos grupos, considerando-se os isolados estudados. Valores percentuais de "bootstrap" são mostrados nos ramos.…”

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae

“…GRSPaV is widely distributed among commercial grape varieties and rootstocks and is associated with several diseases, namely rupestris stem pitting, vein necrosis and Syrah decline (Martelli, 1993;Meng & Gonsalves, 2007). GRSPaV comprises a family of genetic variants (Meng et al, 1999(Meng et al, , 2006Nolasco et al, 2006;Terlizzi et al, 2010Terlizzi et al, , 2011. To date, the genomes of 11 GRSPaV isolates have been sequenced (Meng et al, 1998(Meng et al, , 2005Zhang et al, 1998;Lima et al, 2006Lima et al, , 2009Morelli et al, 2011).…”