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Genome sequence, prevalence and quantification of the first iflavirus identified in a phytoplasma insect vector

Abstract

The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CY) and an efficient vector of flavescence dorée phytoplasma (FD) under laboratory conditions. During a transcriptome sequencing (RNA-seq) project aimed at investigating the interactions between the insect and the two phytoplasmas, a 10,616-nucleotide-long contig with high sequence similarity to known picorna-like viruses was identified among the assembled insect transcripts. The discovery came totally unexpected, because insects from the laboratory colony did not show any evident symptom that could be related to the presence of a virus. The amino acid sequence, the shape and size of viral particles, and the results of phylogenetic analysis suggest that this virus, named Euscelidius variegatus virus 1 (EVV-1), can be considered a new member of a new species in the genus Iflavirus. EVV-1 was detected in all of the tested insects from the laboratory colony used for RNA-seq, both in phytoplasma-exposed and in non-exposed insects, but the viral load measured in FD-exposed samples was significantly lower than that in non-exposed insects. This result suggests the possible existence of an intriguing cross-talk among insects, endogenous bacteria, and viruses. The identification of two other E. variegatus laboratory colonies that were free of EVV-1 could represent the key to addressing some basic virological issues, e.g., viral replication and transmission mechanisms, and offer the opportunity to use infectious clones to express heterologous genes in the leafhopper and manipulate the expression of endogenous genes by promoting virus-induced gene silencing.

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Acknowledgements

The authors thank Flavio Veratti for the maintenance of the E. variegatus colonies in Turin, Riccardo Lenzi for help in the purification of viral particles, and Xavier Foissac and Rodrigo P. P. Almeida for providing adults of E. variegatus from their laboratory colonies.

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Correspondence to Simona Abbà.

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This work was part of the ‘FitoDigIt’ Project funded by Fondazione Cassa di Risparmio di Torino, Torino (Italy), within the ‘Richieste Ordinarie 2014’ and ‘Richieste Ordinarie 2015’ calls. MR was supported by a fellowship funded by the following grant-making foundations: Fondazione Cassa di Risparmio di Cuneo, Fondazione Cassa di Risparmio di Torino, and Fondazione Cassa di Risparmio di Asti in the framework of the INTEFLAVI project.

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The authors declare that they have no conflict of interest.

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Supplemental Fig. S1

The 5’ UTR of EVV-1. The fifteen 5’-terminal nucleotides that were obtained during the RNA-seq assembly but not confirmed by 5’ RACE PCR are highlighted in grey. The number of reads that mapped to the highlighted region is indicated above the sequence. The number of 5’ RACE PCR clones that mapped to the 5’ UTR is shown below the sequence. The predicted 5’-terminal secondary structure is shown in a box (PDF 57 kb)

Supplemental Fig. S2

Phylogenetic analysis of EVV-1 within the family Iflaviridae based on the whole polyprotein. The EVV-1 polyprotein was aligned to polyproteins from 27 other iflaviruses. EVV-1, which is shown in a box, formed a distinct branch with GNV-1 and SBPV. Phylogeny was inferred by the maximum-likelihood method using the CIPRES Science Gateway V. 3.3. Bootstrap analyses with 1000 iterations were used with the Dayhoff substitution matrix to estimate pairwise distances. Only bootstrap values higher than 70 are shown. The scale bar indicates the evolutionary distance for 0.8 amino acid substitutions per site. The taxonomic position of each iflavirus host is indicated by a color code. Iflaviruses that are not yet officially included in the family Iflaviridae by the ICTV are indicated with an asterisk. Abbreviations and GenBank accession numbers of iflaviruses are reported in the legend to Fig. 2 (PDF 478 kb)

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Abbà, S., Galetto, L., Vallino, M. et al. Genome sequence, prevalence and quantification of the first iflavirus identified in a phytoplasma insect vector. Arch Virol 162, 799–809 (2017). https://doi.org/10.1007/s00705-016-3158-3

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Keywords

  • Varroa Destructor
  • Deform Wing Virus
  • Polymerase Chain Reaction Step
  • Covert Infection
  • Ectoparasitic Mite Varroa Destructor