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Two novel Alphaflexiviridae members revealed by deep sequencing of the Vanilla (Orchidaceae) virome

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Abstract

The genomes of two novel viruses were assembled from 454 pyrosequencing data obtained from vanilla leaves from La Réunion. Based on genome organization and homologies, one agent was unambiguously classified as a member of the genus Potexvirus and named vanilla virus X (VVX). The second one, vanilla latent virus (VLV), is phylogenetically close to three unclassified members of the family Alphaflexiviridae with similarity to allexiviruses, and despite the presence of an additional 8-kDa open reading frame, we propose to include VLV as a new member of the genus Allexivirus. Both VVX and VLV were mechanically transmitted to vanilla plants, resulting in asymptomatic infections.

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Acknowledgements

The authors wish to thank Claudie Pavis from UMR ASTRO for smooth and proficient coordination of the Safe-PGR project, the SCIAM Platform at the University of Angers (France) for facilitating the electron microscopy work of AS, and the CRB Vatel and Plantation Vanille Roulof for supplying vanilla material. The technical assistance with lab work by Katia Jade, Alycia Pierret and Jeanne André is kindly acknowledged.

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Authors and Affiliations

  1. Cirad, UMR PVBMT, 97410, Saint Pierre, La Réunion, France

    Michel Grisoni

  2. UMR 1332 BFP, INRA, Univ. Bordeaux, 33882, Villenave d’Ornon, France

    Armelle Marais, Chantal Faure, Sébastien Theil, Sandy Contreras & Thierry Candresse

  3. Cirad, UMR BGPI, 34398, Montpellier, France

    Denis Filloux, Charlotte Julian & Philippe Roumagnac

  4. Anses, LSV, 49044, Angers, France

    Anne Saison

  5. Cirad, UMR AGAP, 97130, Capesterre-Belle-Eau, Guadeloupe, France

    Pierre-Yves Teycheney

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  1. Michel Grisoni
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  2. Armelle Marais
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  3. Denis Filloux
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  11. Thierry Candresse
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Corresponding author

Correspondence to Michel Grisoni.

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Funding

This work was co-funded by the European Union: EraNet-Netbiome program (Safe-PGR project) and European Regional Development Fund (ERDF), by the Conseil Régional de La Réunion, by the French National Research Agency, and by the Centre de Coopération internationale en Recherche agronomique pour le Développement (CIRAD).

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The authors declare that they have no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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705_2017_3540_MOESM1_ESM.jpg

Supplementary Figure1 A) Genomic structure of VLV compared to that of three unassigned members of the Alphaflexiviridae, BVE, ApV and AVS. UTR, untranslated region; RdRp, RNA-dependent RNA polymerase; TBG, triple gene block protein; P40, 40kDa protein; CP, coat protein. B) Alignment of the conserved motif Cx(4)TGHx(6)C) detected in the putative ORF4/ORFx protein of unclassified members of the family Alphaflexiviridae. EscV, escobaria virus (KF421919); WAMV, white ash mosaic virus (GU906791) (JPEG 157 kb)

705_2017_3540_MOESM2_ESM.jpg

Supplementary Figure 2 RNA-dependent RNA polymerase (RdRp) and coat protein (CP) maximum-likelihood trees for VLV and related viruses. Trees were inferred using the Poisson correction model in MEGA 7.0. Clade robustness was assessed by the percent of clustering for 500 bootstrapped trees. The scale bar indicates the number of aa substitutions along the branches. Percentages on the right indicate the range of amino acid sequence identity between species, and within and between genera or groups (JPEG 173 kb)

705_2017_3540_MOESM3_ESM.jpg

Supplementary Figure 3 RNA-dependent RNA polymerase (RdRp) and coat protein (CP) maximum-likelihood trees for VVX and related members of the genus Potexvirus. Trees were inferred using the Poisson correction model in MEGA 7.0. Clade robustness was assessed by the percentage of clustering for 500 bootstrapped trees. The scale bar indicates the number of aa substitutions along the branches. PVX, potato virus X; CymMV, cymbidium mosaic virus; YMV, yam virus X (JPEG 135 kb)

705_2017_3540_MOESM4_ESM.jpg

Supplementary Figure 4 Transmission electron microscopy images of leaf dip preparations from leaves of V. planifolia CR2150 and CR2169 co-infected with VLV and VVX and free of cymbidium mosaic virus (genus Potexvirus) and odontoglossum ringspot virus (genus Tobamovirus). Red bar = 50 nm (JPEG 383 kb)

705_2017_3540_MOESM5_ESM.jpg

Supplementary Figure 5 Spatial distribution of VLV- (red), VVX- (blue) and VLV+VVX- (purple) infected plots (3 plants per plot) in the three shade houses of BRC Vatel (BPL1 to 3) indexed by SDT-RT-PCR in December 2016. Plots testing negative for VLV and VVX are in pale green, areas in white correspond to plots that were vacant at the time of indexing. Plots BPL1-D08 and BPL2-G02 were planted in 2004 and 2006, respectively, with vines introduced from Saint André and presumably co-infected with VLV+VVX. BPL3 was planted in 2013 with vines originating from BPL1 and BPL2 (JPEG 387 kb)

705_2017_3540_MOESM6_ESM.jpg

Supplementary Figure 6 Maximum-likelihood trees based on partial sequences of the CP gene (nt 6685-6911) amplified with primer pair VLV3/VLV4. Phylogenetic trees were inferred using the GTR + G + I model. Bootstraps values (500 replicates) are indicated for clades with more than 50% support. Isolate names are suffixed by their sampling location (shade house number or field location). Arrows connect isolates sharing a common origin by vegetative propagation (dotted line) or mechanical inoculation (solid line) of vines. The scale bar indicates the number of nt substitutions along the branches (JPEG 116 kb)

Supplementary material 7 (XLSX 9 kb)

Supplementary material 8 (XLSX 10 kb)

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Grisoni, M., Marais, A., Filloux, D. et al. Two novel Alphaflexiviridae members revealed by deep sequencing of the Vanilla (Orchidaceae) virome. Arch Virol 162, 3855–3861 (2017). https://doi.org/10.1007/s00705-017-3540-9

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