Abstract
During the last decades new leafroll associated viruses (GLRaVs) have been isolated from grapevine. As a result GLRaVs were raised to 9 while two new isolates (temporary designations GLRaV-Pr and GLRaV-De) were recently determined from Greek grapevine varieties. The majority of these viruses are classified within the recently established genus Ampelovirus of the family Closteroviridae. Based on phylogenies of partial HSP70h sequences, GLRaV-4, -5, -6, -9, -Pr and -De were found to be more closely related to each other compared to the other GLRaVs. This virus group that includes most of the GLRaVs is barely studied, with problems on their characterization, taxonomy and detection. In this study the evolutionary relationships of this virus group were studied based on molecular variability, positive selection analysis and maximum likelihood (ML) phylogenies. Additional sequences from different isolates were determined and datasets corresponding to the N terminal HSP70h and full CP genes we ...
During the last decades new leafroll associated viruses (GLRaVs) have been isolated from grapevine. As a result GLRaVs were raised to 9 while two new isolates (temporary designations GLRaV-Pr and GLRaV-De) were recently determined from Greek grapevine varieties. The majority of these viruses are classified within the recently established genus Ampelovirus of the family Closteroviridae. Based on phylogenies of partial HSP70h sequences, GLRaV-4, -5, -6, -9, -Pr and -De were found to be more closely related to each other compared to the other GLRaVs. This virus group that includes most of the GLRaVs is barely studied, with problems on their characterization, taxonomy and detection. In this study the evolutionary relationships of this virus group were studied based on molecular variability, positive selection analysis and maximum likelihood (ML) phylogenies. Additional sequences from different isolates were determined and datasets corresponding to the N terminal HSP70h and full CP genes were analysed. ML topologies established the closer phylogenetic relationships of GLRaV-4, -5, -6, -9, -Pr and GLRaV-De in regard to the other ampeloviruses, revealing very low inter-species evolutionary distances. The HSP70h phylogeny and bootstrap analysis enabled the identification of species providing a useful taxonomic tool for their rapid demarcation. dN/dS estimations showed that, within the Closteroviridae, these viruses are subjected to the strongest constraints against amino acid substitutions demonstrating a distinct evolutionary trait for this lineage probably related to its particular niche that involves successful adaptation to the host, transmission through vegetative propagation and lack of highly efficient vectors. The HSP70h data set obtained in this study was used for designing new primers, which were applied in nested PCR assays, for the generic detection of members of this lineage and the specific diagnosis of each virus-species. The developed detection scheme is proposed as a tool that can be used in routine screening and certification schemes and could be useful for the enrichment of sequence information of known and novel ampeloviruses, classified within this lineage, enabling their selective amplification in mixed Closteroviridae virus infections. The genomes of GLRaV-Pr and GLRaV-De were further determined using isolated viral dsRNA. GLRaV-Pr is the first fully sequenced grapevine virus of subgroup I ampeloviruses. Its nucleotide sequence is 13,696 nucleotides long, one of the smallest in the Closteroviridae, and contains 7 ORFs which potentially encode a 253 kDa polyprotein, a 58,2 kDa RdRp, a 5,2 kDa protein (p5), a 58,5 kDa HSP70h, a 60 kDa protein (p60), a 30 kDa CP and a protein of 23 kDa (p23). A specific polyclonal antibody was raised against the recombinant GLRaV-Pr CP and was evaluated in different serological assays. A 4,319 nucleotides region corresponding to the HSP70h, p60, CP and p23 (partial) genes was also determined for GLRaV-De. GLRaV-Pr and -De differ by more than 10% in amino acids from the known closely related species. This along with their serological differentiation, genome organization and phylogenetic analysis support their future assignment as tentative species. Comparisons of GLRaV-Pr with the only available genetically related fully sequenced PMWaV-1, PBNSPaV and the partially sequenced GLRaV-9 revealed that, within the genetically diverse genus of ampeloviruses, this lineage of closely related species exhibits high uniformity on genome organization including the smallest and simplest viruses within the Closteroviridae. This observation coupled by Maximum likelihood and Bayesian analysis topologies of the most evolutionary conserved HEL, RdRp and HSP70h proteins obtained herein, confirm that these species follow a distinct evolutionary course and are substantially differentiated from the other virus species classified within the genus Ampelovirus. Consequently, this lineage could in the future constitute a new separate genus. Finally, studies were carried out to eliminate GLRaV-Pr and GRSPaV (Flexiviridae) in two wine cultivars, Mantilaria and Prevezaniko. Virus elimination was achieved by combining in vitro thermotherapy and shoot tip culture. The results confirmed that virus elimination in grapevine is easier to occur on species of the Closteroviridae family than on GRSPaV.
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