Arch Virol (2017) 162:1819–1831 DOI 10.1007/s00705-017-3268-6

VIROLOGY DIVISION NEWS

Capulavirus and Grablovirus: two new genera in the family Geminiviridae

1,2 3 4 5 Arvind Varsani • Philippe Roumagnac • Marc Fuchs • Jesu´s Navas-Castillo • 5 6 7 8 Enrique Moriones • Ali Idris • Rob W. Briddon • Rafael Rivera-Bustamante • 9 10 F. Murilo Zerbini • Darren P Martin

Received: 20 January 2017 / Accepted: 27 January 2017 / Published online: 17 February 2017 Ó Springer-Verlag Wien 2017

Abstract Geminiviruses are plant-infecting single-stran- the treehopper species Spissistilus festinus is the likely ded DNA viruses that occur in most parts of the world. vector of Grapevine red blotch virus. In addition, two Currently, there are seven genera within the family Gem- highly divergent groups of viruses found infecting citrus iniviridae (Becurtovirus, Begomovirus, Curtovirus, Era- and mulberry plants have been assigned to the new species grovirus, Mastrevirus, Topocuvirus and Turncurtovirus). Citrus chlorotic dwarf associated virus and Mulberry The rate of discovery of new geminiviruses has increased mosaic dwarf associated virus, respectively. These species significantly over the last decade as a result of new have been left unassigned to a genus by the ICTV because molecular tools and approaches (rolling-circle amplifica- their particle morphology and insect vectors are unknown. tion and deep sequencing) that allow for high-throughput workflows. Here, we report the establishment of two new genera: Capulavirus, with four new species (Alfalfa leaf Introduction curl virus, Euphorbia caput-medusae latent virus, French bean severe leaf curl virus and Plantago lanceolata latent The family Geminiviridae includes plant-infecting viruses virus), and Grablovirus, with one new species (Grapevine with circular ssDNA genomes that are encapsidated in red blotch virus). The aphid species Aphis craccivora has virions comprised of 22 pentameric capsomeres arranged been shown to be a vector for Alfalfa leaf curl virus, and in a *18- to 30-nm twinned icosahedral (or geminate)

& Arvind Varsani 5 Instituto de Hortofruticultura Subtropical y Mediterra´nea ‘‘La [email protected] Mayora’’, Universidad de Ma´laga-Consejo Superior de ´ & Investigaciones Cientıficas (IHSM-UMA-CSIC), Darren P Martin 29750 Algarrobo-Costa, Ma´laga, Spain [email protected] 6 School of Plant Sciences, University of Arizona, Tucson, 1 The Biodesign Center for Fundamental and Applied AZ 85721-0107, USA Microbiomics, Center for Evolution and Medicine and 7 National Institute for Biotechnology and Genetic Engineering School of Life Sciences, Arizona State University, (NIBGE), Faisalabad, Pakistan Tempe 85287-5001, AZ, USA 8 2 Departamento de Ingenierı´a Gene´tica, Centro de Structural Biology Research Unit, Department of Clinical Investigacio´n y de Estudios Avanzados del IPN (Cinvestav), Laboratory Sciences, University of Cape Town, Unidad Irapuato, 36821 Irapuato, GTO, Mexico Cape Town 7701, South Africa 9 3 Dep. de Fitopatologia/Bioagro, Universidade Federal de CIRAD-INRA-SupAgro, UMR BGPI, Campus International Vic¸osa, Vic¸osa, Minas Gerais 36570-900, Brazil de Montferrier-Baillarguet, 34398 Montpellier Cedex-5, France 10 Computational Biology Group, Institute of Infectious 4 Diseases and Molecular Medicine, University of Cape Town, Section of Plant Pathology and Plant-Microbe Biology, Cape Town 7925, South Africa School of Integrative Plant Science, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA

123 1820 A. Varsani et al. configuration [7, 41]. Geminiviruses have a near-global Fig. 1 Genome organisation of viruses in the genera of the familyc distribution and infect both monocotyledonous and Geminiviridae. Citrus chlorotic dwarf associated virus and Mulberry mosaic dwarf associated virus have been accepted by the Executive dicotyledonous plants. Geminiviruses are often responsible Committee of the International Committee on Taxonomy of Viruses for serious yield losses in economically important crops, (ICTV) as a species within the family Geminiviridae but have not including tomato, maize, cotton, chickpea and cassava been assigned to a genus. AGmV and GGVA have not yet been [25]. Symptoms of geminivirus infections can include considered as representatives of a species by ICTV foliar crinkling, curling, yellowing, stunting, mosaic and/or striations. in Hungary, Israel, Japan and South Korea (tentatively The Geminiviridae Study Group of the International referred to as grapevine geminivirus A; GGVA) sharing Committee on Taxonomy of Viruses (ICTV) has provided [97% pairwise identity have been discovered [2]. These a series of guidelines for the classification of viruses within have distinctly geminivirus-like genome organisations and the seven currently recognised genera of the family: Be- are more closely related to members of some established curtovirus, Begomovirus, Curtovirus, Eragrovirus, Mas- geminivirus species than they are to citrus chlorotic dwarf trevirus, Topocuvirus and Turncurtovirus. The criteria associated virus (CCDaV) and mulberry mosaic dwarf include host range, insect vector, genome organisation and associated virus (MMCaV). The taxonomic assignment of genome-wide pairwise sequence identities AGmV and GGVA still needs to be discussed by the ICTV. [1, 11, 12, 26, 38, 39]. Whereas the arrangements of genes and intergenic Over the last decade, the development of new molecular regions in the genomes of curtoviruses, topocuviruses, tools such as rolling-circle amplification (RCA) and high- turncurtoviruses, AGmV, GGVA and genomes/DNA A throughput sequencing have greatly facilitated the discov- components of begomoviruses are all very similar, the ery of novel geminiviruses in a range of cultivated and non- becurtoviruses, capulaviruses, eragroviruses, grabloviruses cultivated host species [29]. Accordingly, these techniques and mastreviruses all have distinctly unique genome have accelerated the discovery of highly divergent gemi- organisations (Fig. 1). Furthermore, the genome organisa- nivirus-like viruses. For some of these viruses, a large tion of CCDaV is also apparently unique [21]. Particularly number of closely related full genome sequences have been noteworthy in the CCDaV genome (3640 nt) is the *921- determined. Analyses of these sequences (pairwise identi- nt-long virion-sense ORF, V3, which results in the CCDaV ties and phylogenetics), coupled with the identification of genome being 600-800 nt larger than those of the other insect vectors and particle morphology, have allowed us to known monopartite geminiviruses (Fig. 1). determine with great confidence that some of these diver- Although the genomes of the known geminiviruses can gent viruses should be classified as members of two new have up to eight genes, only two of them, the replication- genera in the family Geminiviridae, which was approved associated protein gene (rep) and the coat protein gene (cp) by the Executive Committee of the ICTV: are obviously conserved across viruses in all of the genera, whereas the replication enhancer gene (ren), symptom 1) Capulavirus, which will for now include four determinant gene (sd) and silencing suppressor gene (ss) species, Alfalfa leaf curl virus, Euphorbia caput- homologues are found in begomoviruses, curtoviruses, medusae latent virus, French bean severe leaf curl topocuviruses and turncurtoviruses (Fig. 1), and transcrip- virus and Plantago lanceolata latent virus tion activator gene (trap) homologues are additionally [5, 6, 30, 36] found in eragroviruses (Fig. 1). The genomes of viruses in 2) Grablovirus, which will for now include one species, all of the established genera also have either proven or Grapevine red blotch virus [18, 28, 35] putative movement protein genes (mp), which, in the In addition, two other species of highly divergent viruses monopartite members of the family, are immediately that are closely related to geminiviruses have been accep- upstream of cp. It is currently unknown, however, if these ted by the Executive Committee of the ICTV for inclusion genes in viruses from different genera have a common in the family Geminiviridae and will remain unassigned to evolutionary origin, because the amino acid sequences that a genus, awaiting identification of the vector species and they encode have such low degrees of similarity that it is confirmation of particle morphology. These new species not possible to conclude that they are homologous. are: The genomes of becurtoviruses, capulaviruses, gra- bloviruses, eragroviruses, mastreviruses, and CCDaVs all 1) Citrus chlorotic dwarf associated virus [17, 21] have two intergenic regions (Fig. 1). In general, the longer 2) Mulberry mosaic dwarf associated virus [23, 24] of these is referred to as the long intergenic region (LIR) Furthermore, recently, a single virus isolated from an and contains a conserved nonanucleotide within a hairpin apple tree in China (tentatively referred to as apple gemi- structure that forms the origin of virion-strand replication nivirus; AGmV) [20] and 13 virus isolates from grapevine [3, 22]. Similarly, the shorter intergenic region is referred 123 The genera Capulavirus and Grablovirus 1821

Begomoviruses

V2 C4 AC4 CRA CRB

Monopartite DNA-A AV1 DNA-B BV1 (TYLCV) V1 (BGMV) (BGMV) C1 BC1 AC1 C3 AC3

Bipartite C2 AC2

V3 V2 C3 V3 V2 C4 V4 V3 C1 V4:V2 Becurtoviruses Capulaviruses Curtoviruses (BCTIV) C1 (BCTV) C1:C2 C1 V1 C1:C2 V1 V1 C3

C2

V2 V2 C3 V2

C1 V1 C1 Grabloviruses Eragroviruses Mastreviruses (ECSV) V1 (MSV) C1:C2 C1 C1:C2 V1

V3 C2 C2

Unclassified

V2 C4 C4 V2 V2 C4 LIR SIR CR Topocuviruses Turncurtoviruses C1 GGVA C1 (TPCTV) (TCTV) V1 V1 V1 nsp C1 cp

C3 C3 mp possible mp C2 C2 C2 reg

Unassigned rep repA C1 V3 C4 V2 ren V2 V2 trap/ss C1 possible trap/ss C1:C2 C1 CCDaV V1 MMCaV AGmV sd C1:C2 V1 possible sd V1 possible reg C3 unknown V3 V4 C2

123 1822 A. Varsani et al. to as the short intergenic region (SIR) and contains tran- Fig. 2 A. Unrooted neighbour-joining tree inferred from aligned full-c scription termination signals and, for mastreviruses at least, genome sequences of representative isolates from various genera in the family Geminiviridae. Numbers associated with branches indicate the origin of complementary-strand replication. percentage bootstrap support for these branches (determined with Introns are present in several geminivirus genes. Rep 1000 bootstrap replicates). B. Maximum-likelihood phylogenetic tree and RepA proteins in becurtoviruses, capulaviruses, gra- (applying the WAG?I?G amino acid substitution model) inferred bloviruses, mastreviruses, MMCaV and CCDaV are either from aligned CP sequences of representative isolates from various genera in the family Geminiviridae. Numbers associate with branches proven to be, or inferred to be, expressed from alternatively indicate percentage aLRT support for these branches. The CP spliced complementary-sense transcripts [8, 13, 40] phylogenetic tree is rooted at the midpoint. Branches with less than (Fig. 1). Further, an intron occurs in the mp of mastre- 80% aLRT support have been collapsed. C. Maximum-likelihood viruses [40], and an intron has been inferred to occur in the phylogenetic tree (applying the LG?I?G amino acid substitution model) inferred from aligned Rep sequences of representative isolates mp of the capulavirus EcmLV (Fig. 1)[5]. from various genera in the family Geminiviridae. Numbers associated Recombination has played a significant role in the with branches indicate percentage aLRT support for these branches. evolution of geminiviruses, and it can therefore be difficult The Rep phylogenetic tree is rooted with the Rep sequences of to accurately infer the evolutionary relationships of viruses members of the family Genomoviridae (not included here). Branches with less than 80% aLRT support have been collapsed in different genera. Whereas non-homologous recombina- tion has potentially contributed to the loss or gain of large genome regions in particular genera, homologous recom- bination has also likely contributed to the exchange of possibly the products of inter-genus recombination events genome regions between viruses in different genera [10, 37]. For example, the becurtovirus CPs are most clo- [6, 9, 31, 33]. sely related to those of curtoviruses, whereas the becur- Nonetheless, we inferred the approximate genome-wide tovirus Reps are most closely related to those of CCDaV evolutionary relationships between representative members and MMCaV (Fig. 2 and Fig. 3). of the various established geminivirus genera, CCDaV, MMCaV, AGmV and GGVA (Fig. 1). The full genome Capulavirus sequences of these representative viruses were aligned using MUSCLE [15] and used to infer a neighbor-joining A group of closely related viruses isolated from Euphorbia phylogenetic tree with the Jukes-Cantor nucleotide substi- caput-medusae (South Africa), Medicago sativa (France tution model and 1000 bootstrap replicates. Branches with and Spain), Phaseolus vulgaris (India) and Plantago \60% support were collapsed using TreeGraph2 [34], and lanceolata (Finland) have been assigned to the new genus the tree was rooted at the midpoint (Fig. 2). From this Capulavirus. The genus name Capulavirus was derived phylogenetic tree, it is apparent that the capulaviruses and from the type member of the genus: euphorbia caput-me- grabloviruses are closely related to one another and that dusae latent virus. As with viruses in the genera Mastre- CCDaV and MMCaV are closely related to one another. virus and Becurtovirus, capulaviruses have two intergenic We also analysed the evolutionary relationships of the regions and express Rep from a spliced complementary- inferred CP and Rep amino acid sequences of the same set strand transcript. In common with begomoviruses and of representative geminiviruses. The CP and Rep sequence curtoviruses, they have a large complementary-sense ORF datasets were aligned using MUSCLE [15] and used to (C3) that is completely embedded within rep. A unique infer maximum-likelihood phylogenetic trees using feature of capulavirus genomes is a complex arrangement PHYML3.0 [16], applying the WAG?I?G and LG?I?G of possible MP-encoding ORFs located in the 5’direction substitution model, respectively (selected as the best-fit from cp (Fig. 1). Two or more of these ORFs may con- models by ProtTest [14]; Fig. 2). Branch support in these stitute an intron-containing mp (Fig. 1). Geminate particles trees was determined using approximate likelihood ratio have been observed in purified preparations of EcmLV by tests (aLRT). The Rep phylogenetic tree was rooted with transmission electron microscopy [30]. Aphids of the spe- sequences from the family Genomoviridae [19], whereas cies Aphis craccivora have been shown to transmit ALCV the CP tree was rooted at the midpoint. Branches with [30]. All known capulaviruses have the nonanucleotide \80% aLRT support were collapsed using TreeGraph2 motif ‘‘TAATATTAC’’ at their presumed origins of virion- [34]. Pairwise CP and Rep amino acid sequence identities strand replication. were determined using SDT v1.2 [27] (Fig. 3). An analysis of the distribution of pairwise identities (one Discordance in the phylogenetic placement of cur- minus Hamming distances of pairwise aligned sequences toviruses, becurtoviruses, topocuviruses, eragroviruses, with pairwise deletion of gaps) of known capulavirus turncurtoviruses and CCDaV in the CP and Rep phyloge- genomes using SDT v1.2 [27](n= 47; Table 1) indicates netic trees likely reflects the fact that the most recent that a pairwise-identity-based species demarcation criterion common ancestors of the known viruses in these genera are that would minimize conflicts (i.e., possible assignments of 123 The genera Capulavirus and Grablovirus 1823

Genome Genera 100 KP410285 Beet curly top Iran virus 100 HQ443515 Spinach curly top Arizona virus Becurtovirus 100 U49907 Horseradish curly top virus 100 GU734126 Spinach severe curly top virus AF379637 Beet curly top virus Curtovirus 100 EU921828 Pepper yellow dwarf virus 100 KC108902 Turnip curly top virus 100 GU456685 Turnip curly top virus Turncurtovirus 100 KT388088 Turnip leaf roll virus 100 KT388086 Turnip leaf roll virus 94 KM386645 Apple geminivirus unclassified KX618694 Grapevine geminivirus A X84735 Tomato pseudo-curly top virus Topocuvirus 100 100 FM877473 African cassava mosaic virus JF502373 Cotton leaf curl Burewala virus Begomovirus 100 FJ665283 Bean golden mosaic virus 100 KC706535 Sida micrantha mosaic virus FJ665634 Eragrostis curvula streak virus Eragrovirus 100 FJ665630 Eragrostis curvula streak virus EF536860 Wheat dwarf virus DQ458791 Chickpea chlorotic dwarf virus 93 KJ437671 Axonopus compressus streak virus Mastrevirus 68 AF003952 Maize streak virus 100 KF147918 Grapevine red blotch virus JX559642 Grapevine red blotch virus Grablovirus 100 JQ920490 Citrus chlorotic dwarf associated virus unclassified 89 KR131749 Mulberry crinkle-associated virus KT214389 Plantago lanceolata latent virus 67 JX094280 French bean severe leaf curl virus 100 KT214373 Alfalfa leaf curl virus Capulavirus 100 KT214386 Euphorbia caput-medusae latent virus 0.1 nucleotide substitutions per site

CP 88 AF379637 Beet curly top virus EU921828 Pepper yellow dwarf virus 97 U49907 Horseradish curly top virus Curtovirus GU734126 Spinach severe curly top virus 99 KP410285 Beet curly top Iran virus 97 HQ443515 Spinach curly top Arizona virus Becurtovirus 91 GU456685 Turnip curly top virus 100 KC108902 Turnip curly top virus Turncurtovirus 88 92 KT388086 Turnip leaf roll virus KT388088 Turnip leaf roll virus 93 AF003952 Maize streak virus KJ437671 Axonopus compressus streak virus 92 DQ458791 Chickpea chlorotic dwarf virus Mastrevirus 98 EF536860 Wheat dwarf virus 100 FJ665630 Eragrostis curvula streak virus 96 FJ665634 Eragrostis curvula streak virus Eragrovirus X84735 Tomato pseudo-curly top virus Topocuvirus 95 JX094280 French bean severe leaf curl virus 89 KT214373 Alfalfa leaf curl virus 100 KT214386 Euphorbia caput-medusae latent virus Capulavirus KT214389 Plantago lanceolata latent virus 100 KM386645 Apple geminivirus unclassified KX618694 Grapevine geminivirus A JX559642 Grapevine red blotch virus Grablovirus 100 KF147918 Grapevine red blotch virus 99 JQ920490 Citrus chlorotic dwarf associated virus unclassified KR131749 Mulberry crinkle-associated virus 85 FJ665283 Bean golden mosaic virus 99 KC706535 Sida micrantha mosaic virus Begomovirus 99 FM877473 African cassava mosaic virus JF502373 Cotton leaf curl Burewala virus 0.5 amino acid substitutions per site 91

Rep 100 GU456685 Turnip curly top virus 96 KC108902 Turnip curly top virus Turncurtovirus 100 KT388086 Turnip leaf roll virus 99 KT388088 Turnip leaf roll virus AF379637 Beet curly top virus Curtovirus EU921828 Pepper yellow dwarf virus 100 KX618694 Grapevine geminivirus A unclassified 96 KM386645 Apple geminivirus 94 FM877473 African cassava mosaic virus 91 JF502373 Cotton leaf curl Burewala virus Begomovirus 96 X84735 Tomato pseudo-curly top virus Topocuvirus FJ665283 Bean golden mosaic virus 91 Begomovirus 100 100 KC706535 Sida micrantha mosaic virus GU734126 Spinach severe curly top virus Curtovirus 100 U49907 Horseradish curly top virus 100 FJ665630 Eragrostis curvula streak virus FJ665634 Eragrostis curvula streak virus Eragrovirus 100 JX559642 Grapevine red blotch virus KF147918 Grapevine red blotch virus Grablovirus KT214389 Plantago lanceolata latent virus 84 KT214386 Euphorbia caput-medusae latent virus 100 KT214373 Alfalfa leaf curl virus Capulavirus 91 JX094280 French bean severe leaf curl virus 82 EF536860 Wheat dwarf virus 90 AF003952 Maize streak virus 100 KJ437671 Axonopus compressus streak virus Mastrevirus DQ458791 Chickpea chlorotic dwarf virus 97 JQ920490 Citrus chlorotic dwarf associated virus unclassified 82 KR131749 Mulberry crinkle-associated virus HQ443515 Spinach curly top Arizona virus 99 Becurtovirus 100 KP410285 Beet curly top Iran virus 0.2 amino acid substitutions per site

123 1824 A. Varsani et al. individual isolates to two or more species) could be placed Analysis of the 27 known grablovirus full-genome either in the 72 to 84% identity interval, or in the 87-94% sequences indicate that they all share [91% genome-wide interval (Fig. 4). Whereas a species demarcation threshold nucleotide sequence identity (Fig. 5) and are therefore all within the 87-94% range would yield five species, placing assigned to the species Grapevine red blotch virus. All of it within the 72-84% range would yield four species. In the the GRBV isolates share \45% Rep amino acid sequence case of a threshold within the 87-94% range, the alfalfa leaf identity with all viruses in other geminivirus genera curl virus (ALCV) sequences, which have all been isolated (Fig. 3). Regardless of whether the full-genome nucleotide from alfalfa plants, would be split into two species. Given sequence, Rep amino acid sequence or CP amino acid is that there seems to be no good biological reason to split the considered, the GRBV isolates cluster together with 100% ALCVs into two species, we have opted to tentatively aLRT support (Fig. 2). place the species demarcation threshold in the 72-84% In the absence of additional related sequences in the range. To bring the capulavirus species demarcation genus, we suggest that, based on the species demarcation threshold in line with that of the genus Mastrevirus (the criteria used for most other geminivirus genera, grablovirus recognized genus of geminiviruses that the capulaviruses isolates sharing\80% genome-wide pairwise identity with are most closely related to; Fig. 4) we have opted to ten- members of accepted grablovirus species should be clas- tatively select 78% as the pairwise identity value above sified as members of distinct grablovirus species. This is a which two sequences should be considered isolates of the tentative species demarcation criterion that will need to be same species. The four proposed capulavirus species to refined as more full-genome sequences of grabloviruses are which the 47 known capulavirus full genome determined. sequences (Table 1) have been assigned are Alfalfa leaf curl virus (n = 26), Euphorbia caput-medusae latent virus (n = 17), French bean severe leaf curl virus (n = 2) and Plantago lanceolata latent virus (n = 2). Based on pair- New geminivirus species that remain unassigned wise identity comparisons, all isolates (ALCV; EcmLV; to a genus french bean severe leaf curl virus, FbSLCV; plantago lanceolata latent virus, PlLV) within each of these species Citrus chlorotic dwarf associated virus and Mulberry share between 63% and 73% genome-wide sequence mosaic dwarf associated virus identity with all isolates that have been assigned to other proposed capulavirus species (Fig. 2). Two complete CCDaV genomes, isolated from citrus trees Regardless of whether the full-genome nucleotide in Turkey (JQ920490) [21] and China (KF561253), and sequence, the inferred Rep amino acid sequence, or the eight complete MMCaV genomes, isolated from mulberry inferred CP amino acid sequence is considered, all of the bushes in China (KP303687, KP699128-KP699132, viruses belonging to the proposed capulavirus species KP728254, KR131749) [23, 24], are clearly closely related group with 100% aLRT support with other proposed to those of geminiviruses, based on both their geminivirus- capulaviruses (Fig. 2). The viruses assigned to the genus like genome organization and the fact that their inferred Capulavirus share \22% CP amino acid sequence identity Rep and CP amino acid sequences cluster phylogenetically and \45% Rep amino acid sequence identity with viruses with those of the known geminiviruses (Fig. 1 and Fig. 2). in other established genera of the family Geminiviridae All known CCDaV and MMDaV isolates also have the (Fig. 3). nonanucleotide TAATATTAC, which is characteristic of the virion-strand origin of replication of the majority of Grablovirus geminiviruses. The two CCDaV sequences share 99.5% genome-wide A group of closely related viruses discovered infecting pairwise identity, whereas the eight MMDaV sequences grapevines in Canada, South Korea and the USA have been share [97% identity. The predicted Rep proteins of these assigned to the genus Grablovirus. The genus name is CCDaV and MMDaV isolates share *58% amino acid based on the name of the type member: grapevine red sequence similarity with one another, but only 33-42% blotch virus (GRBV), the first and currently the only similarity with those of other geminiviruses (Fig. 3). member of this genus [18]. These viruses have the virion- Regardless of whether full genome nucleotide sequences, strand origin of replication nonanucleotide motif TAA- inferred Rep amino acid sequences, or inferred CP TATTAC and a unique genome arrangement (Fig. 1; amino acid sequences are considered, the two proposed Table 2). The natural vector is likely the three-cornered species group separately from all other geminiviruses, alfalfa treehopper (Spissistilus festinus Say) [4]. with 100% aLRT branch support (Fig. 2).

123 The genera Capulavirus and Grablovirus 1825 Mastrevirus Curtovirus Capulavirus Becurtovirus Turncurtovirus Topocuvirus Begomovirus Unclassified Eragrovirus Grablovirus

CP

Rep Sida micrantha mosaic virus [KC706535] Eragrostis curvula streak virus [FJ665630] Eragrostis curvula streak virus [FJ665634] Arizona virus [HQ443515] Spinach curly top Grapevine red blotch virus [KF147918] Turnip curly top virus [KC108902] Turnip French bean severe leaf curl virus [JX094280] Euphorbia caputmedusae latent virus [KT214386] Plantago lanceolata latent virus [KT214389 ] Wheat dwarf virus [EF536860] Beet curly top virus [AF379637] Beet curly top virus [EU921828] Spinach severe curly top virus [GU734126] Cotton leaf curl Burewala virus [JF502373] Citrus chlorotic dwarf associated virus [JQ920490] Alfalfa leaf curl virus [KT214373] Maize streak virus [AF003952] Axonopus compressus streak virus [KJ437671] Chickpea chlorotic dwarf virus [DQ458791] Horseradish curly top virus [U49907] Beet curly top Iran virus [KP410285] African cassava mosaic virus [FM877473] Mulberry mosaic dwarf associated virus [KP303687] Turnip curly top virus [GU456685] Turnip leaf roll virus [KT388086] Turnip leaf roll virus [KT388088] Turnip Tomato pseudocurly top virus [X84735] Tomato Bean golden mosaic virus [FJ665283] Grapevine geminivirus A [KX618694] Grapevine red blotch virus [JX559642] Apple geminivirus [KM386645] French bean severe leaf curl virus [JX094280] 78 72 48 24 26 25 23 26 26 25 26 24 23 24 24 24 23 23 24 21 26 24 20 24 24 24 22 23 19 22 Capulavirus Alfalfa leaf curl virus [KT214373] 75 75 47 22 23 24 23 26 25 25 24 22 20 22 24 25 25 26 23 23 23 24 20 24 25 26 23 23 21 24 Euphorbia caputmedusae latent virus [KT214386] 80 77 53 23 24 24 25 24 24 24 24 24 24 22 24 22 22 24 25 22 24 23 20 23 26 23 24 23 20 20 Plantago lanceolata latent virus [KT214389 ] 69 68 73 24 24 24 24 26 26 25 25 24 24 24 23 27 28 22 21 23 21 21 23 22 25 22 24 24 24 24 Maize streak virus [AF003952] 39 44 40 38 39 35 36 34 34 28 28 26 25 28 25 28 28 28 28 27 21 21 19 20 22 23 21 21 22 25 Mastrevirus Axonopus compressus streak virus [KJ437671] 41 43 44 43 46 35 34 35 35 24 24 22 23 22 22 28 28 26 27 23 20 19 23 20 21 24 22 25 25 24 Wheat dwarf virus [EF536860] 39 40 40 39 47 47 36 45 44 24 28 29 29 29 26 24 24 26 29 26 22 21 19 20 16 23 20 19 25 24 Chickpea chlorotic dwarf virus [DQ458791] 44 45 45 44 49 51 48 35 35 29 29 29 28 31 30 25 26 27 26 24 20 22 23 24 19 24 23 27 22 23 Eragrovirus Eragrostis curvula streak virus [FJ665630] 39 43 39 42 31 36 32 35 98 27 27 29 25 26 26 27 27 27 27 28 21 22 20 21 17 21 19 21 26 26 Eragrostis curvula streak virus [FJ665634] 39 42 40 42 31 37 32 35 91 27 27 29 23 26 26 29 29 27 28 28 21 23 21 21 17 21 19 21 26 26 Beet curly top virus [AF379637] 38 38 40 36 32 36 37 36 42 43 93 81 82 85 79 41 41 42 41 23 25 29 24 28 24 30 14 24 23 23 Curtovirus Beet curly top virus [EU921828] 36 38 37 37 34 37 34 37 43 42 66 84 82 84 78 40 40 41 41 24 25 29 23 28 22 29 19 24 24 23 Horseradish curly top virus [U49907] 42 42 41 40 33 34 35 37 45 46 56 56 77 82 76 44 44 40 43 24 25 28 23 28 23 27 18 23 25 23 Spinach severe curly top virus [GU734126] 42 45 44 40 35 39 36 38 46 47 56 55 79 86 82 45 45 42 43 24 26 26 26 26 24 28 20 22 24 23 Becurtovirus Spinach curly top Arizona virus [HQ443515] 34 37 34 35 32 36 31 38 31 31 31 31 29 34 84 45 45 43 42 24 25 29 27 27 23 28 15 21 24 24 Beet curly top Iran virus [KP410285] 33 37 34 37 33 36 33 38 32 31 31 32 29 33 86 41 41 40 40 23 27 27 26 27 22 29 19 26 24 21 Turnip curly top virus [GU456685] 37 40 40 39 33 36 35 34 42 41 62 65 49 49 32 32 99 79 77 28 21 20 24 25 24 24 26 27 27 26 Turncurtovirus Turnip curly top virus [KC108902] 37 40 40 39 33 36 35 35 41 40 63 66 49 49 32 32 93 81 77 28 21 19 24 21 23 24 24 27 27 26 Turnip leaf roll virus [KT388086] 36 38 38 38 33 37 37 37 41 41 64 66 53 53 33 31 76 75 96 24 23 22 21 20 23 23 18 24 24 26 Turnip leaf roll virus [KT388088] 39 41 42 38 36 37 34 36 42 42 64 59 53 55 33 30 68 66 75 24 20 20 22 20 20 23 21 24 24 24 Topocuvirus Tomato pseudocurly top virus [X84735] 39 39 40 36 35 38 36 38 45 45 71 65 60 59 33 32 62 61 65 62 21 24 23 24 21 23 19 22 23 20 Bean golden mosaic virus [FJ665283] 38 39 40 38 30 36 34 35 44 44 64 63 59 59 30 28 58 57 63 59 75 93 75 70 31 29 17 19 23 22 Sida micrantha mosaic virus [KC706535] 37 37 37 36 30 37 31 36 42 42 67 62 60 57 31 29 60 59 64 60 76 80 75 71 31 29 19 19 23 24 Begomovirus Cotton leaf curl Burewala virus [JF502373] 38 39 41 38 34 36 37 35 41 42 66 58 52 52 30 29 56 56 58 60 70 67 67 74 29 29 18 22 23 24 African cassava mosaic virus [FM877473] 38 41 39 37 35 36 36 35 44 44 60 57 52 52 34 32 59 57 60 57 65 63 65 79 32 30 17 21 25 24 Citrus chlorotic dwarf associated virus [JQ920490] 37 39 40 39 35 40 37 39 38 37 36 34 33 35 40 38 39 40 40 38 39 37 36 36 37 38 21 23 21 19 Mulberry mosaic dwarf associated virus [KP303687] 39 42 42 40 35 41 37 37 39 39 37 36 33 38 42 42 35 36 34 39 36 34 34 35 33 53 21 27 26 27 Unclassified Apple geminivirus [KM386645] 40 40 40 39 34 37 34 35 46 45 57 55 52 50 30 29 55 54 56 54 63 59 59 68 64 37 34 48 25 24 Grapevine geminivirus A [KX618694] 36 36 35 34 33 35 32 34 45 45 58 57 49 53 30 30 57 55 55 57 60 59 60 62 62 33 35 60 24 25 Grapevine red blotch virus [JX559642] 44 45 44 44 39 38 36 39 38 39 38 40 33 40 31 29 36 37 38 39 39 37 37 38 39 39 40 35 38 96 Grablovirus Grapevine red blotch virus [KF147918] 44 45 44 45 39 39 36 40 38 38 39 41 34 40 31 32 37 38 39 39 40 37 38 38 39 39 40 35 39 91

Pairwise identity (%) 23 31 57 74 83 14 40 48 66 91 100

Fig. 3 Pairwise identities of the Rep and CP sequences of representative isolates from various genera in the family Geminiviridae as determined using SDT v1.2 (Muhire et al., 2014)

123 1826 A. Varsani et al.

Table 1 Details of virus isolates that have been assigned to the four proposed species within the genus Capulavirus Capulavirus Accession no. Virus acronym Isolate Country Host

Alfalfa leaf curl virus KP732474 ALCV 44-1E France Medicago sativa KT214350 ALCV TDV14_44-16 France Medicago sativa KT214351 ALCV PB14_LUZ166 France Medicago sativa KT214352 ALCV ASS14_Assas2 France Medicago sativa KT214353 ALCV BON14_LUZ075 France Medicago sativa KT214354 ALCV SSL14_Toul1 France Medicago sativa KT214355 ALCV ALB14_LUZ147 France Medicago sativa KT214356 ALCV ALB14_LUZ163 France Medicago sativa KT214357 ALCV SSL14_Toul7 France Medicago sativa KT214358 ALCV TDV12_48-2A France Medicago sativa KT214359 ALCV PB14_LUZ184 France Medicago sativa KT214360 ALCV ASS14_Assas1 France Medicago sativa KT214361 ALCV VAU14_LUZ136 France Medicago sativa KT214362 ALCV GAG14_LUZ193 France Medicago sativa KT214363 ALCV PB14_LUZ182 France Medicago sativa KT214364 ALCV ALB14_LUZ148 France Medicago sativa KT214365 ALCV PB14_LUZ-GS4 France Medicago sativa KT214366 ALCV ALB14_LUZ164 France Medicago sativa KT214367 ALCV PB14_LUZ171 France Medicago sativa KT214368 ALCV PB14_LUZ188 France Medicago sativa KT214369 ALCV PB14_LUZ-GS6 France Medicago sativa KT214370 ALCV BON14_LUZ076 France Medicago sativa KT214371 ALCV PB14_LUZ165 France Medicago sativa KT214372 ALCV PB14_LUZ178 France Medicago sativa KT214373 ALCV VAU14_LUZ142 France Medicago sativa KT214374 ALCV PB14_GS8 France Medicago sativa KT214375 ALCV PB14_LUZ179 France Medicago sativa Euphorbia caput-medusae latent virus HF921459 EcmLV Dar10 South Africa Euphorbia caput-medusae HF921460 EcmLV Dar11 South Africa Euphorbia caput-medusae HF921477 EcmLV Lap11 South Africa Euphorbia caput-medusae KT214376 EcmLV DAR12_CM243 South Africa Euphorbia caput-medusae KT214377 EcmLV DAR12_CM26 South Africa Euphorbia caput-medusae KT214378 EcmLV DAR12_CM176 South Africa Euphorbia caput-medusae KT214379 EcmLV DAR12_CM27 South Africa Euphorbia caput-medusae KT214380 EcmLV DAR12_CM192 South Africa Euphorbia caput-medusae KT214381 EcmLV DAR12_CM186 South Africa Euphorbia caput-medusae KT214382 EcmLV DAR12_CM240 South Africa Euphorbia caput-medusae KT214383 EcmLV DAR12_CM162 South Africa Euphorbia caput-medusae KT214384 EcmLV PAT12_CM96 South Africa Euphorbia caput-medusae KT214385 EcmLV PAT12_CM99 South Africa Euphorbia caput-medusae KT214386 EcmLV PAT12_CM101 South Africa Euphorbia caput-medusae KT214387 EcmLV DAR12_CM217 South Africa Euphorbia caput-medusae KT214388 EcmLV DAR12_CM251 South Africa Euphorbia caput-medusae French bean severe leaf curl virus JX094280 FbSLCV FbSLCV-1 India Phaseolus vulgaris JX094281 FbSLCV FbSLCV-2 India Phaseolus vulgaris Plantago lanceolata latent virus KT214389 PlLV ALA13_Pl11 Finland Plantago lanceolata KT214390 PlLV ALA13_Pl5 Finland Plantago lanceolata

123 eemnduigSTv. [ v1.2 SDT using determined 7koncplvrsgenomes. capulavirus known 47 A. 4 Fig. genera The B A

0.05 nucleotidesubstitutionspersite Proportion of percentage pairwise identities 87 0.00 0.05 0.10 0.15 0.20 itiuino ariencetd euneiette of identities sequence nucleotide pairwise of Distribution 60 Percentage identity Capulavirus 100 61 100 62 100 99 63 100 71 65 100 64 100 100 and 100 99 100 95 65 77 100 100 27 FbSLCV [JX094281] FbSLCV [JX094280] Grablovirus EcmLV [HF921477] EcmLV [HF921460] EcmLV [HF921459] EcmLV [KT214384] EcmLV [KT214386] EcmLV [KT214385] EcmLV [KT214388] EcmLV [KT214380] EcmLV [KT214382] EcmLV [KT214376] EcmLV [KT214379] EcmLV [KT214377] EcmLV [KT214383] EcmLV [KT214387] EcmLV [KT214381] EcmLV [KT214378] 66 ALCV [KP732474] ALCV [KT214372] ALCV [KT214370] ALCV [KT214361] ALCV [KT214360] ALCV [KT214368] ALCV [KT214359] ALCV [KT214375] ALCV [KT214363] ALCV [KT214362] ALCV [KT214358] ALCV [KT214366] ALCV [KT214364] ALCV [KT214371] ALCV [KT214367] ALCV [KT214369] ALCV [KT214365] ALCV [KT214373] ALCV [KT214351] ALCV [KT214374] ALCV [KT214350] ALCV [KT214356] ALCV [KT214355] ALCV [KT214357] ALCV [KT214354] ALCV [KT214352] ALCV [KT214353] n egbrjiigphylogenetic neighbor-joining a and ] PlLV [KT214390] PlLV [KT214389] B 67 eoewd arieidentities pairwise Genome-wide . 68 69 70

FbSLCV [JX094280] 71 FbSLCV [JX094281] ALCV [KP732474] 72

ALCV [KT214353] 73 ALCV [KT214352] ALCV [KT214354] 74

ALCV [KT214357] 75 ALCV [KT214355]

ALCV [KT214356] 76 ALCV [KT214350] ALCV [KT214374] 77

ALCV [KT214351] 78 ALCV [KT214373] ALCV [KT214365] 79 reo auaiu slts h rei otdwt mastrevirus with rooted is tree The shown) (not isolates. sequences genome capulavirus of tree

ALCV [KT214369] 80 ALCV [KT214367] ALCV [KT214371] 81

ALCV [KT214364] 82 ALCV [KT214366] ALCV [KT214358] 83

ALCV [KT214362] 84 ALCV [KT214363] ALCV [KT214375] 85 ALCV [KT214359] ALCV [KT214368] 86

ALCV [KT214360] 87 ALCV [KT214361] ALCV [KT214370] 88

ALCV [KT214372] 89 EcmLV [HF921459] EcmLV [KT214378] 90

EcmLV [KT214381] 91 EcmLV [KT214387] EcmLV [HF921460] 92

EcmLV [KT214383] 93 EcmLV [KT214377] EcmLV [KT214379] 94

EcmLV [KT214376] 95 EcmLV [KT214382]

EcmLV [KT214380] 96 EcmLV [KT214388] EcmLV [KT214385] 97 123

EcmLV [KT214386] Pairwise identity (%) 98 EcmLV [HF921477] EcmLV [KT214384] 99 1827 100 PlLV [KT214389] 63 67 70 74 78 81 85 89 93 96 100 PlLV [KT214390] 1828 A. Varsani et al.

Table 2 Details of virus isolates that have been assigned to the proposed species Grapevine red blotch virus within the genus Grablovirus Grablovirus Accession no. Virus acronym Isolate Country Host

Grapevine red blotch virus KF147916 GRBV NY175 USA1 Vitis vinifera KU564249 GRBV NY701 USA2 Vitis vinifera cv.Cabernet Sauvignon JQ901105 GRBV JRT[456]17NOV10 USA3 Vitis vinifera KC896623 GRBV CF214-1 USA Vitis vinifera KC896625 GRBV Z1A-1 USA Vitis vinifera KF137562 GRBV OR1a USA4 Vitis vinifera cv. Pinot noir KF147917 GRBV NY135 USA5 Vitis vinifera KF147918 GRBV NY137 USA5 Vitis vinifera KF751708 GRBV NY147 USA3 Vitis vinifera cv. Pinot Noir KP221559 GRBV 1 USA Vitis vinifera cv. Early Burgundy KU564247 GRBV NY699 USA2 Vitis californica x Vitis vinifera hybrid KU564248 GRBV NY700 USA2 Vitis californica x Vitis vinifera hybrid KU564250 GRBV NY702 USA2 Vitis vinifera cv. Merlot KU564251 GRBV NY703 USA2 Vitis vinifera cv. Cabernet franc KU564252 GRBV NY704 USA2 Vitis vinifera cv. Cabernet franc KU564253 GRBV NY705 USA2 Vitis vinifera cv. Cabernet franc KU564254 GRBV NY913 USA2 Vitis californica x Vitis vinifera hybrid KU564255 GRBV NY921 USA2 Vitis vinifera cv. Cabernet franc KU564256 GRBV NY926 USA2 Vitis vinifera cv. Cabernet franc JX559642 GRBV 3138-03 Canada Vitis vinifera KC896624 GRBV CS337-1 USA Vitis vinifera KF147915 GRBV NY271 USA Vitis vinifera KU821056 GRBV SW6 South Korea Vitis vinifera KC427993 GRBV GiGV-WA-RS USA Vitis vinifera KC427994 GRBV GiGV-WA-DS USA Vitis vinifera KC427995 GRBV GiGV-WA-MR USA Vitis vinifera cv. Merlot KC427996 GRBV GiGV-WA-CF USA Vitis vinifera cv. Cabernet franc US state: 1MD, 2CA, 3NY, 4OR, 5PA

CCDaV and MMDaV will need to remain unassigned to (KX570607, KX570609, KX570614) group together, with a genus until their respective vector species are identified 94% bootstrap support (Fig. 2). These viruses isolated from and/or it is confirmed that they form geminate particles. woody plants contain the typical geminivirus-like nanonucleotide motif TAATATTAC and display a dis- tinctly geminivirus-like genome organisation. In addition, these viruses are clearly recombinant, with a begomovirus- New geminiviruses identified recently that need like Rep (sharing 59-68% amino acid sequence identity) to be reviewed by ICTV and a divergent CP that is unlike that of any known geminivirus. Apple geminivirus and grapevine geminivirus A The thirteen GGVA sequences share[97% identity. The unique AGmV sequence and the thirteen GGVA sequences One AGmV complete genome isolated from an apple tree share *63% genome-wide pairwise identity. The Rep and CP in China (accession no. KM386645) [20] and thirteen sequences of the AGmV and GGVA isolates share *60% and GGVA complete genomes isolated from grapevine in 48% amino acid sequence identity, respectively, with one China (KX570611), Hungary (KX570618), Israel another. The Rep proteins are most closely related to those of (KX618694), Japan (KX570610, KX570612, KX570613, begomoviruses, curtoviruses, topocuvirus and turncur- KX570615, KX570616, KX570617) and South Korea toviruses, sharing 50-68% identity (Fig. 3). Although, based

123 The genera Capulavirus and Grablovirus 1829

A 0.5

0.4

0.3

0.2

0.1

0.0 Proportion of percentage pairwise identities 90 91 92 93 94 95 96 97 98 99 100 Percentage identity

B GBRV [JX559642] GBRV [JQ901105] 100 GBRV [KC896625] GBRV [KC896623] 99 GBRV [KU564255] 98 100 GBRV [KF137562] GBRV [KF751708] 97 GBRV [KP221559] 96 GBRV [KU564247] Pairwise identity (%) GBRV [KU564248] 96 95 GBRV [KU564250] 95 GBRV [KU564254] GBRV [KU564251] 94 GBRV [KU564252] 93 GBRV [KU564253] GBRV [KU564256] 92 GBRV [KC427993] 91 GBRV [KC427994] GBRV [KC427995] GBRV [KC427996] GBRV [KF147916] GBRV [KF147915] 86 GBRV [KU564249] GBRV [KC896624] 98 GBRV [KU821056]

100 GBRV [KF147917] GBRV [KF147918]

0.01 nucleotide substitutions per site GBRV [JX559642] GBRV [KF147916] GBRV [KF147917] GBRV [KF137562] GBRV [KF147915] GBRV [KF147918] GBRV [KF751708] GBRV [JQ901105] GBRV [KP221559] GBRV [KU564250] GBRV [KU564252] GBRV [KU564256] GBRV [KC427993] GBRV [KU564253] GBRV [KC427994] GBRV [KC427995] GBRV [KC427996] GBRV [KC896625] GBRV [KU564247] GBRV [KU821056] GBRV [KU564254] GBRV [KU564251] GBRV [KU564249] GBRV [KC896624] GBRV [KC896623] GBRV [KU564248] GBRV [KU564255]

Fig. 5 A. Distribution of pairwise nucleotide sequence identities of of grablovirus isolates. The tree is rooted with mastrevirus genome 27 grablovirus genomes. B. Genome-wide pairwise identities deter- sequences (not shown) mined using SDT v1.2 [27] and a neighbor-joining phylogenetic tree on inferred Rep, AGmV and GGVA branch with bego- Concluding remarks moviruses, both viruses group separately from all other geminiviruses, with 100% aLRT branch support (Fig. 2), The past ten years have seen the establishment of five new based on the inferred CP amino acid sequences. geminivirus genera, and the next ten will likely see many AGmV and GGVA must also remain unassigned to a more being established as metagenomics-based approaches genus until the vectors are identified and/or geminate par- begin enabling the convenient and cost-effective discovery ticles are observed. of viruses in greater numbers from cultivated and non-

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