Arch Virol DOI 10.1007/s00705-017-3587-7
ANNOTATED SEQUENCE RECORD
Determination of the complete genomic sequence of grapevine virus H, a novel vitivirus infecting grapevine Thierry Candresse1 · Sébastien Theil1 · Chantal Faure1 · Armelle Marais1
Received: 27 July 2017 / Accepted: 21 September 2017 © Springer-Verlag GmbH Austria 2017
Abstract The present work reports the discovery and the complete genome sequencing of a novel member of the genus Vitivirus in the family Betaflexiviridae (subfamily Trivirinae) from a symptomless grapevine of unknown variety from Portugal. Total RNAs extracted from phloem scrapings were sequenced using Illumina technology. Bioinformatic analysis of the RNA-seq data revealed a mixed infection involving three viruses and two viroids in addition to a novel vitivirus. Completion and analysis of the genome sequence (7446 nt excluding the polyA tail) showed a typical vitivirus genomic organization. Phylogenetic analysis of the various ORFs clearly showed the new virus to belong in the genus Vitivirus, but sequence divergence firmly establishes it as a member of a new species, for which the name “Grapevine virus H” is proposed. Vitivirus is a genus containing plant-infecting viruses in the family Betaflexiviridae (subfamily Trivirinae) of the order Tymovirales [1]. Members of this genus are characterized by a genomic organization consisting of five open reading frames (ORFs) with an unusual ORF encoding a protein of ca. 20 kDa of unknown function, located
Handling Editor: Sead Sabanadzovic. The nucleotide sequence reported in this work has been deposited in the GenBank database under accession number MF521889. * Thierry Candresse [email protected] 1
UMR 1332 Biologie du Fruit et Pathologie, INRA, Univ. Bordeaux, CS 20032, 33882 Villenave d’Ornon Cedex, France
immediately downstream of ORF1, which encodes the viral replicase (REP) [1, 2]. Expression of all downstream ORFs probably occurs through a set of overlapping subgenomic messenger RNAs [3]. According to current taxonomy [4], the known vitiviruses comprise grapevine viruses A, B, D E and F (reviewed in ref.5), actinidia viruses A and B [6], and heracleum latent virus and mint virus 2 [7]. At least three recently described novel viruses, arracacha virus V [8], Agave tequilana leaf virus (GenBank NC_034833), and grapevine virus K (GenBank NC_035202), are likely genus members. Several vitiviruses have been shown to be transmitted by pseudococcid mealybugs and/or scale insects (GVA, GVB, GVE) [5, 9] or by aphids (HLV, MV-2) [1]. In grapevine, several vitiviruses have been associated with syndromes of the rugose wood complex [5, 9]. GVA is the putative agent of the grapevine Kober stem grooving syndrome and is also tentatively associated with Shiraz disease. GVB is associated with grapevine corky bark and GVD, with corky rugose wood, while GVF could be responsible for graft incompatibility problems in cv. Cabernet Sauvignon [5, 9]. No specific symptoms are known to be associated with GVE infection [5, 9]. The new vitivirus was identified in a grapevine (cv. unknown) collected in Portugal that did not show any specific viral infection symptoms. Total nucleic acid was extracted from phloem scrapings using the protocol of Xiao et al. [10]. Extracted total RNAs were converted to cDNA (TruSeq Stranded Total Library Kit, Illumina) and used for Illumina multiplexed sequencing (2 × 150 bp paired reads, Hi-Seq3000 sequencer). After demultiplexing and quality trimming, a total of 2,125,051 high-quality reads were obtained for the grapevine sample and assembled using CLC Genomics Workbench 9.0 with standard settings or
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T. Candresse et al.
Fig. 1 Schematic representation of the genome organization of grapevine virus H. The various open reading frames are indicated by rectangular boxes. REP, replicase; MP, movement protein; CP, capsid protein; NABP, nucleic acid binding protein; A(n), polyA 3’ tail
Fig. 2 Neighbor-joining phylogenetic trees reconstructed using the amino acid sequences of the replicase (A) and capsid protein (B) of Vitivirus genus members. Strict amino acid sequence identity distances were used for construction of the tree, and the statistical significance of branches was evaluated by bootstrap analysis (1,000
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replicates). Only values higher than 70% are indicated. The scale bar represents 10% amino acid sequence divergence (A) or 5% aa sequence divergence (B). For some viruses, only partial sequences are available and were used. Grapevine virus H proteins are indicated in bold and indicated by a black triangle
Complete genomic sequence of grapevine virus H
an in-house-developed pipeline integrating the Newbler assembler and the obtained contigs, which were annotated using BlastN and BlastX by comparison with sequences in the GenBank database. This revealed a mixed infection involving grapevine leafroll-associated virus 3, grapevine rupestris stem pitting virus, grapevine fleck virus, and hop stunt and grapevine yellow speckle 1 viroids. In addition, two contigs of 4.57kb (minimal coverage 53x) and 1.14 kb (minimal coverage 100x), respectively, showing similarity to various vitivirus sequences were also observed. Further analysis [11] of the sequence data allowed the reconstruction of a ca. 7.3-kb scaffold, integrating 0.6% of the total reads (average scaffold coverage, 241x). The 5’ end of the genome was determined using total RNA as template for 5’ rapid amplification of cDNA ends (RACE, Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France), while the 3’ end of the genome was determined by RT-PCR using a polyT primer and an internal primer designed from the scaffold sequence. The complete genome sequence of the new agent is 7446 nucleotides (nt) long (GenBank MF521889), which is a typical size for a vitivirus genome [1]. It shows the typical vitivirus genomic organization with five ORFs (Fig. 1), and in particular contains an ORF2 putatively coding for a ca. 20-kDa product, which is characteristic of members of this genus. All ORFs also have length typical of those of Vitivirus genus members [1, 5, 9]. The genome contains 5’ and 3’ non-coding regions (NCR) of 97 and 85 nt, respectively, excluding the 3’ poly(A) tail. ORF1, which encodes the viral replicase (REP, 195.1 kDa), shows highest sequence similarity to its AVB counterpart (54.4% nt sequence identity; 50.9% aa sequence identity), followed by that of GVB (53.1% nt sequence identity; 49.1% aa sequence identity). It contains all of the conserved motifs that are typical of these proteins in Betaflexiviridae family members. A phylogenetic analysis conducted on the REP sequence confirmed these relationships and showed the new virus to fall clearly within the genus Vitivirus (Fig. 2A). A similar analysis performed on the capsid protein (CP, 21.5 kDa) encoded by ORF4 showed that it was most closely related to that of GVD (65.0% nt and 66.5% aa sequence identity), followed by the recent GVK sequence (63.8% nt and 63.3% aa sequence identity). This was confirmed by phylogenetic analysis of the CP sequence (Fig. 2B). For both the RdRp and the CP, these values are well below the 72% nt and 80% aa sequence identity thresholds separating species in the family Betaflexiviridae [1], clearly demonstrating that the novel agent should be considered as a representative of a new Vitivirus species, for which the name “Grapevine virus H” is proposed. The other ORF products were also most closely related to those of other genus Vitivirus members: ORF3 (movement protein, MP [12], 29.2 kDa) yielded the highest BlastP score with the GVA3 MP (46%
aa sequence identity); and ORF5 (NABP, silencing suppressor activity [13, 14], 12.2 kDa), with the MV-2 NABP (56% identity). The product of ORF2 (18.0 kDa) did not appear to be related to any of the proteins available in the GenBank database. The grapevine plant that was the source of GVH did not display any obvious disease symptoms despite being infected by a total of three viruses and two viroids in addition to GVH. Clearly, the absence of symptoms and the mixed infection status of the original host preclude drawing any tentative conclusions on the potential pathogenicity of GVH. A small-scale survey performed in the plot were the original plant was collected indicated that the virus was present in six of the 20 plants tested, again without any correlation with any symptomatology. Further studies are therefore needed to evaluate this specific point as well as other important questions, such as the potential existence and identity of GVH vectors in the field. Acknowledgements The authors thank the Platform Genotoul (INRA, Toulouse, France) for the Illumina sequencing. Compliance with ethical standards Conflict of interest All authors (TC, ST, CF and AM) declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.
References 1. Adams MJ, Candresse T, Hammond J, Kreuze JF, Martelli GP, Namba S, Pearson MN, Ryu KH, Saldarelli P, Yoshikawa N (2012) Family Betaflexiviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy. 9th Report of the ICTV. Elsevier Academic Press, Amsterdam, The Netherlands, pp 920–941 2. Martelli GP, Minafra A, Saldarelli P (1997) Vitivirus, a new genus of plant viruses. Arch Virol 142:1929–1932 3. Galiakparov N, Goszczynski DE, Che X, Batuman O, BarJoseph M, Mawassi M (2003) Two classes of subgenomic RNA of GVA produced by internal controller elements. Virology 312:434–448 4. ICTV (2017) ICTV Master species list 2016 v1.3. https://talk. ictvonline.org/files/master-species-lists/m/msl/6776 5. Minafra A, Mawassi M, Goszczynski D, Saldarelli P (2017) Grapevine Vitiviruses. In: Meng B, Martelli GP, Golino DA, Fuchs M (eds) Grapevine viruses: molecular biology, diagnostics and management. Springer, Cham, pp 229–256 6. Blouin AG, Chavan RR, Pearson MN, MacDiarmid RM, Cohen D (2012) Detection and characterisation of two novel vitiviruses infecting Actinidia. Arch Virol 157:713–722 7. Tzanetakis IE, Postman JD, Martin RR (2007) Identification, detection and transmission of a new vitivirus from Mentha. Arch Virol 152:2027–2033
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8. Oliveira LM, Orílio AF, Inoue-Nagata AK, Nagata T, Blawid R (2017) A novel vitivirus-like sequence found in Arracacia xanthorrhiza plants by high throughput sequencing. Arch Virol 162:2141–2144 9. Martelli GP (2014) Rugose wood complex. J. Plant Pathol 96(1S):73–88 10. Xiao H, Kim WS, Meng B (2015) A highly effective and versatile technology for the isolation of RNAs from grapevines and other woody perennials for use in virus diagnostics. Virol J 12:171. doi:10.1186/s12985-015-0376-3 11. Candresse T, Marais A, Faure C, Gentit P (2013) Association of Little cherry virus 1 with the Shirofugen Stunt Disease and genome characterization of a divergent LChV1 isolate. Phytopathology 103:293–298
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T. Candresse et al. 12. Haviv S, Moskovitz Y, Mawassi M (2012) The ORF-3 encoded proteins of vitiviruses GVA and GVB induce tubule-like and punctate structures during virus infections and localize to plasmodesmata. Virus Res 163:291–301 13. Zhou ZS, Dell’Orco M, Saldarelli P, Turturo M, Minafra A, Martelli GP (2006) Identification of an RNA silencing suppressor in the genome of Grapevine virus A. J Gen Virol 87:2387–2395 14. Chiba M, Reed JC, Prokhnevsky AI, Chapman EJ, Mawassi M, Koonin EV, Carrington JC, Dolja VV (2006) Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon. Virology 346:7–14
ANNOTATED SEQUENCE RECORD
Determination of the complete genomic sequence of grapevine virus H, a novel vitivirus infecting grapevine Thierry Candresse1 · Sébastien Theil1 · Chantal Faure1 · Armelle Marais1
Received: 27 July 2017 / Accepted: 21 September 2017 © Springer-Verlag GmbH Austria 2017
Abstract The present work reports the discovery and the complete genome sequencing of a novel member of the genus Vitivirus in the family Betaflexiviridae (subfamily Trivirinae) from a symptomless grapevine of unknown variety from Portugal. Total RNAs extracted from phloem scrapings were sequenced using Illumina technology. Bioinformatic analysis of the RNA-seq data revealed a mixed infection involving three viruses and two viroids in addition to a novel vitivirus. Completion and analysis of the genome sequence (7446 nt excluding the polyA tail) showed a typical vitivirus genomic organization. Phylogenetic analysis of the various ORFs clearly showed the new virus to belong in the genus Vitivirus, but sequence divergence firmly establishes it as a member of a new species, for which the name “Grapevine virus H” is proposed. Vitivirus is a genus containing plant-infecting viruses in the family Betaflexiviridae (subfamily Trivirinae) of the order Tymovirales [1]. Members of this genus are characterized by a genomic organization consisting of five open reading frames (ORFs) with an unusual ORF encoding a protein of ca. 20 kDa of unknown function, located
Handling Editor: Sead Sabanadzovic. The nucleotide sequence reported in this work has been deposited in the GenBank database under accession number MF521889. * Thierry Candresse [email protected] 1
UMR 1332 Biologie du Fruit et Pathologie, INRA, Univ. Bordeaux, CS 20032, 33882 Villenave d’Ornon Cedex, France
immediately downstream of ORF1, which encodes the viral replicase (REP) [1, 2]. Expression of all downstream ORFs probably occurs through a set of overlapping subgenomic messenger RNAs [3]. According to current taxonomy [4], the known vitiviruses comprise grapevine viruses A, B, D E and F (reviewed in ref.5), actinidia viruses A and B [6], and heracleum latent virus and mint virus 2 [7]. At least three recently described novel viruses, arracacha virus V [8], Agave tequilana leaf virus (GenBank NC_034833), and grapevine virus K (GenBank NC_035202), are likely genus members. Several vitiviruses have been shown to be transmitted by pseudococcid mealybugs and/or scale insects (GVA, GVB, GVE) [5, 9] or by aphids (HLV, MV-2) [1]. In grapevine, several vitiviruses have been associated with syndromes of the rugose wood complex [5, 9]. GVA is the putative agent of the grapevine Kober stem grooving syndrome and is also tentatively associated with Shiraz disease. GVB is associated with grapevine corky bark and GVD, with corky rugose wood, while GVF could be responsible for graft incompatibility problems in cv. Cabernet Sauvignon [5, 9]. No specific symptoms are known to be associated with GVE infection [5, 9]. The new vitivirus was identified in a grapevine (cv. unknown) collected in Portugal that did not show any specific viral infection symptoms. Total nucleic acid was extracted from phloem scrapings using the protocol of Xiao et al. [10]. Extracted total RNAs were converted to cDNA (TruSeq Stranded Total Library Kit, Illumina) and used for Illumina multiplexed sequencing (2 × 150 bp paired reads, Hi-Seq3000 sequencer). After demultiplexing and quality trimming, a total of 2,125,051 high-quality reads were obtained for the grapevine sample and assembled using CLC Genomics Workbench 9.0 with standard settings or
13
Vol.:(0123456789)
T. Candresse et al.
Fig. 1 Schematic representation of the genome organization of grapevine virus H. The various open reading frames are indicated by rectangular boxes. REP, replicase; MP, movement protein; CP, capsid protein; NABP, nucleic acid binding protein; A(n), polyA 3’ tail
Fig. 2 Neighbor-joining phylogenetic trees reconstructed using the amino acid sequences of the replicase (A) and capsid protein (B) of Vitivirus genus members. Strict amino acid sequence identity distances were used for construction of the tree, and the statistical significance of branches was evaluated by bootstrap analysis (1,000
13
replicates). Only values higher than 70% are indicated. The scale bar represents 10% amino acid sequence divergence (A) or 5% aa sequence divergence (B). For some viruses, only partial sequences are available and were used. Grapevine virus H proteins are indicated in bold and indicated by a black triangle
Complete genomic sequence of grapevine virus H
an in-house-developed pipeline integrating the Newbler assembler and the obtained contigs, which were annotated using BlastN and BlastX by comparison with sequences in the GenBank database. This revealed a mixed infection involving grapevine leafroll-associated virus 3, grapevine rupestris stem pitting virus, grapevine fleck virus, and hop stunt and grapevine yellow speckle 1 viroids. In addition, two contigs of 4.57kb (minimal coverage 53x) and 1.14 kb (minimal coverage 100x), respectively, showing similarity to various vitivirus sequences were also observed. Further analysis [11] of the sequence data allowed the reconstruction of a ca. 7.3-kb scaffold, integrating 0.6% of the total reads (average scaffold coverage, 241x). The 5’ end of the genome was determined using total RNA as template for 5’ rapid amplification of cDNA ends (RACE, Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France), while the 3’ end of the genome was determined by RT-PCR using a polyT primer and an internal primer designed from the scaffold sequence. The complete genome sequence of the new agent is 7446 nucleotides (nt) long (GenBank MF521889), which is a typical size for a vitivirus genome [1]. It shows the typical vitivirus genomic organization with five ORFs (Fig. 1), and in particular contains an ORF2 putatively coding for a ca. 20-kDa product, which is characteristic of members of this genus. All ORFs also have length typical of those of Vitivirus genus members [1, 5, 9]. The genome contains 5’ and 3’ non-coding regions (NCR) of 97 and 85 nt, respectively, excluding the 3’ poly(A) tail. ORF1, which encodes the viral replicase (REP, 195.1 kDa), shows highest sequence similarity to its AVB counterpart (54.4% nt sequence identity; 50.9% aa sequence identity), followed by that of GVB (53.1% nt sequence identity; 49.1% aa sequence identity). It contains all of the conserved motifs that are typical of these proteins in Betaflexiviridae family members. A phylogenetic analysis conducted on the REP sequence confirmed these relationships and showed the new virus to fall clearly within the genus Vitivirus (Fig. 2A). A similar analysis performed on the capsid protein (CP, 21.5 kDa) encoded by ORF4 showed that it was most closely related to that of GVD (65.0% nt and 66.5% aa sequence identity), followed by the recent GVK sequence (63.8% nt and 63.3% aa sequence identity). This was confirmed by phylogenetic analysis of the CP sequence (Fig. 2B). For both the RdRp and the CP, these values are well below the 72% nt and 80% aa sequence identity thresholds separating species in the family Betaflexiviridae [1], clearly demonstrating that the novel agent should be considered as a representative of a new Vitivirus species, for which the name “Grapevine virus H” is proposed. The other ORF products were also most closely related to those of other genus Vitivirus members: ORF3 (movement protein, MP [12], 29.2 kDa) yielded the highest BlastP score with the GVA3 MP (46%
aa sequence identity); and ORF5 (NABP, silencing suppressor activity [13, 14], 12.2 kDa), with the MV-2 NABP (56% identity). The product of ORF2 (18.0 kDa) did not appear to be related to any of the proteins available in the GenBank database. The grapevine plant that was the source of GVH did not display any obvious disease symptoms despite being infected by a total of three viruses and two viroids in addition to GVH. Clearly, the absence of symptoms and the mixed infection status of the original host preclude drawing any tentative conclusions on the potential pathogenicity of GVH. A small-scale survey performed in the plot were the original plant was collected indicated that the virus was present in six of the 20 plants tested, again without any correlation with any symptomatology. Further studies are therefore needed to evaluate this specific point as well as other important questions, such as the potential existence and identity of GVH vectors in the field. Acknowledgements The authors thank the Platform Genotoul (INRA, Toulouse, France) for the Illumina sequencing. Compliance with ethical standards Conflict of interest All authors (TC, ST, CF and AM) declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.
References 1. Adams MJ, Candresse T, Hammond J, Kreuze JF, Martelli GP, Namba S, Pearson MN, Ryu KH, Saldarelli P, Yoshikawa N (2012) Family Betaflexiviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy. 9th Report of the ICTV. Elsevier Academic Press, Amsterdam, The Netherlands, pp 920–941 2. Martelli GP, Minafra A, Saldarelli P (1997) Vitivirus, a new genus of plant viruses. Arch Virol 142:1929–1932 3. Galiakparov N, Goszczynski DE, Che X, Batuman O, BarJoseph M, Mawassi M (2003) Two classes of subgenomic RNA of GVA produced by internal controller elements. Virology 312:434–448 4. ICTV (2017) ICTV Master species list 2016 v1.3. https://talk. ictvonline.org/files/master-species-lists/m/msl/6776 5. Minafra A, Mawassi M, Goszczynski D, Saldarelli P (2017) Grapevine Vitiviruses. In: Meng B, Martelli GP, Golino DA, Fuchs M (eds) Grapevine viruses: molecular biology, diagnostics and management. Springer, Cham, pp 229–256 6. Blouin AG, Chavan RR, Pearson MN, MacDiarmid RM, Cohen D (2012) Detection and characterisation of two novel vitiviruses infecting Actinidia. Arch Virol 157:713–722 7. Tzanetakis IE, Postman JD, Martin RR (2007) Identification, detection and transmission of a new vitivirus from Mentha. Arch Virol 152:2027–2033
13
8. Oliveira LM, Orílio AF, Inoue-Nagata AK, Nagata T, Blawid R (2017) A novel vitivirus-like sequence found in Arracacia xanthorrhiza plants by high throughput sequencing. Arch Virol 162:2141–2144 9. Martelli GP (2014) Rugose wood complex. J. Plant Pathol 96(1S):73–88 10. Xiao H, Kim WS, Meng B (2015) A highly effective and versatile technology for the isolation of RNAs from grapevines and other woody perennials for use in virus diagnostics. Virol J 12:171. doi:10.1186/s12985-015-0376-3 11. Candresse T, Marais A, Faure C, Gentit P (2013) Association of Little cherry virus 1 with the Shirofugen Stunt Disease and genome characterization of a divergent LChV1 isolate. Phytopathology 103:293–298
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T. Candresse et al. 12. Haviv S, Moskovitz Y, Mawassi M (2012) The ORF-3 encoded proteins of vitiviruses GVA and GVB induce tubule-like and punctate structures during virus infections and localize to plasmodesmata. Virus Res 163:291–301 13. Zhou ZS, Dell’Orco M, Saldarelli P, Turturo M, Minafra A, Martelli GP (2006) Identification of an RNA silencing suppressor in the genome of Grapevine virus A. J Gen Virol 87:2387–2395 14. Chiba M, Reed JC, Prokhnevsky AI, Chapman EJ, Mawassi M, Koonin EV, Carrington JC, Dolja VV (2006) Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon. Virology 346:7–14
