Virology 426 (2012) 1–6

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A novel class of DNA satellites associated with New World begomoviruses

Elvira Fiallo-Olivé a,b, Yamila Martínez-Zubiaur a, Enrique Moriones b, Jesús Navas-Castillo b,⁎a Centro Nacional de Sanidad Agropecuaria (CENSA), San José de Las Lajas, Mayabeque, Cubab Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), Consejo Superior de Investigaciones Científicas, Estación Experimental “La Mayora”,29750 Algarrobo-Costa, Málaga, Spain

⁎ Corresponding author. Fax: +34 952552677.E-mail address: jnavas@eelm.csic.es (J. Navas-Castill

0042-6822/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.virol.2012.01.024

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 December 2011Returned to author for revision 11January 2012Accepted 18 January 2012Available online 11 February 2012

Keywords:GeminiviridaeBegomovirusesDNA satellitesWeedsMalvaceae

Begomoviruses (family Geminiviridae) are whitefly-transmitted plant DNA viruses that have been shownto be helper viruses for a number of distinct DNA satellites, including betasatellites and alphasatellites.Replication of the satellites interferes to some degree with replication of the helper and in some casesthey affect the disease symptoms. To date, betasatellites and related molecules such as the satellite associ-ated with Tomato leaf curl virus (ToLCV-sat), have only been associated with Old World begomoviruses.Here, we report the discovery and characterization of subviral molecules associated with bipartite begomo-viruses from the New World, which constitute a novel class of DNA satellites, in two malvaceous plantspecies. These molecules, in addition to sharing some genetic features with betasatellites and ToLCV-sat,contain nucleotide stretches of begomoviral origin, presumably the remains of recombination events in-volved in their origin.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Some isolates of certain plant viruses contain nucleic acids otherthan genomic, subgenomic or defective nucleic acids, which lackgenes that would encode the enzymes required for their replication.Two major classes of such satellite agents can be distinguishedaccording to the source of the coat protein used to encapsulate thenucleic acid: the satellite viruses, which code for their own coat pro-tein, and the satellite nucleic acids, which are packaged by the coatprotein of the helper virus. The genetic material of satellite virusesand satellite nucleic acids comprises small molecules that have little,if any, sequence similarity to the helper virus genome. Replication ofthe satellite interferes to some degree with the replication of thehelper virus and in some cases it affects the disease symptoms(Hull, 2002). Although there are numerous satellite RNAs associatedwith RNA plant viruses, as first reported in the 1960s (reviewed byHu et al., 2009), it was not until 1997 that the first DNA satellitewas described: ToLCV-sat, associated with Tomato leaf curl virus(ToLCV), a plant virus in the genus Begomovirus (family Geminiviridae)(Dry et al., 1997).

Members of this family have circular single-stranded DNA ge-nomes encapsidated in twinned quasi-icosahedral (geminate) vi-rions. Most geminiviruses have bipartite genomes and the twosegments (referred to as DNA-A and DNA-B) are similar in size(2.5–3 kb). In contrast, monopartite begomoviruses have only one

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rights reserved.

genome component that is homologous to DNA-A. Geminivirus ge-nomes are replicated by a rolling-circle mechanism initiated by thereplication-associated protein (Rep), the only virus-encoded proteinrequired for replication (Fontes et al., 1994; Stenger et al., 1991).The origin of replication is in a stem–loop structure located in theintergenic region (IR), which contains a conserved nonanucleotidesequence (TAATATTAC) (Orozco and Hanley-Bowdoin, 1996; Stanley,1995). The IR also includes virus-specific iterated sequences up-stream of the origin of replication to which Rep binds, referred to asiterons (Argüello-Astorga and Ruiz-Medrano, 2001; Argüello-Astorgaet al., 1994).

The genus Begomovirus, which currently includes about 200accepted virus species, is by far the largest of the four genera in thefamily Geminiviridae. Over the last 20 years or so, begomoviruseshave emerged as serious constraints to the cultivation of a varietyof crops in various parts of the world but especially in the tropicsand subtropics (Morales, 2010; Navas-Castillo et al., 2011; Rojasand Gilbertson, 2008). Various devastating begomoviruses have alsospread into more temperate regions, where they seriously reducegreenhouse crop production.

The ToLCV satellite (ToLCV-sat) is 682 nt in length and, with theexception of the hairpin structure that is conserved in all membersof the family Geminiviridae, its sequence is unrelated to that ofToLCV, a begomovirus occurring in Australia. This satellite has no dis-cernable effects on either viral replication or on the symptoms causedby ToLCV (Dry et al., 1997). Although it depends on the Rep protein ofToLCV for replication, the Rep binding motif of ToLCV-sat, located in asecondary stem–loop structure, does not appear to strictly fit the re-quirements that dictate the specificity of interaction of geminiviral

2 E. Fiallo-Olivé et al. / Virology 426 (2012) 1–6

Rep proteins with their cognate replication origin, which includes theiterons (Lin et al., 2003). In fact, ToLCV-sat replication is supported bya number of begomoviruses, including Tomato yellow leaf curl virusand African cassava mosaic virus, and even by the curtovirus Beetcurly top virus (Dry et al., 1997).

To date, two additional types of DNA satellites associated withbegomoviruses have been described: betasatellites and alphasatel-lites (Fig. 1). Betasatellites, first referred to as DNA-β, are satellitemolecules that are associated with monopartite begomoviruses andare ~1360 nt in length (approximately half the size of the helpervirus genome). Like ToLCV-sat, betasatellites are unrelated insequence to their helper viruses and are dependent on them for rep-lication, movement in plants and insect transmission. Betasatellitesare widespread in the Old World, where the helper monopartitebegomoviruses are known to occur, and although experimentaltransreplication by New World begomoviruses has been reported(Nawaz-ul-Rehman et al., 2009), they have not yet been found inthe Americas. Betasatellites have a highly conserved organizationconsisting of an adenine-rich region (A-rich), a region that is con-served among all betasatellites (known as the satellite conservedregion [SCR] and also present in ToLCV-sat), and a single openreading frame (ORF) in the complementary strand that codes forthe βC1 protein (Briddon et al., 2003, 2008). The SCR contains apotential hairpin structure with the loop sequence TAA/GTATTAC,similar to that of the origin of replication of geminiviruses and nano-viruses (Briddon et al., 2003). Unlike ToLCV-sat, betasatellites aug-ment the accumulation of their helper begomoviruses and enhancethe symptoms induced in some host plants (Briddon et al., 2001;Nawaz-ul-Rehman and Fauquet, 2009; Patil and Fauquet, 2010;Saunders et al., 2000), probably due to the silencing suppressor ac-tivity of the βC1protein (Cui et al., 2005; Saeed et al., 2005).

In addition to betasatellites, some begomovirus diseases are asso-ciated with an additional single-stranded DNA component, originallynamed DNA-1 and now known as alphasatellites (Briddon et al.,2004). Alphasatellites are approximately half the size of begomoviruscomponents (~1375 nt) and show a common organization consistingof a single ORF coding for a Rep protein with similarity to those ofnanoviruses (Mansoor et al., 1999; Saunders and Stanley, 1999), an

alphasatellites

(~1375 nt)

betasatellites

(~1360 nt) ToLCV-sat (682 nt)

ORF

Stem-loop

SCR

A-rich region

Iterons - TATA

177H1 (687 nt)

Fig. 1. Schematic representation of the DNA satellites previously known to be asso-ciated with begomoviruses (alphasatellites, betasatellites, ToLCV-sat), including themain genome features: ORFs, conserved stem–loop, satellite conserved region [SCR]and A-rich region. The clone 177H1 isolated from M. coromandelianum is includedfor comparison, with indication of the iteron-like region.

A-rich region and a predicted hairpin structure with the sequenceTAGTATTAC (Briddon et al., 2004). Consequently, these molecules,unlike typical satellites, are capable of self-replication in host plantsbut require a helper begomovirus for movement within the plant aswell as for insect transmission (Saunders and Stanley, 1999;Saunders et al., 2000). At least in one case, an unusual class of alpha-satellites has been shown to attenuate begomovirus-betasatellitesymptoms by reducing betasatellite DNA accumulation (Idris et al.,2011). Recently, two distinct alphasatellites have been found to be as-sociated with New World begomoviruses. In Brazil, they were associ-ated with two bipartite begomoviruses infecting weeds (Euphorbiamosaic virus and Cleome leaf crumple virus), and in both cases theycontain the conserved genome features of alphasatellites, includinga gene encoding a Rep protein, an A-rich region and a hairpin struc-ture similar to those of a group of alphasatellites from Africa(Paprotka et al., 2010). The alphasatellite-like molecule found inVenezuela was associated with the bipartite begomovirus Melonchlorotic mosaic virus, and although its sequence diverged from thatof typical Old World alphasatellites, it had all the aforementioned ge-nome features of this type of DNA satellite (Romay et al., 2010).

In this work, we report the discovery and characterization of sub-viral molecules associated with bipartite begomoviruses from theNew World which constitute a novel class of DNA satellites, in twomalvaceous plant species. These molecules, in addition to sharingvarious genetic features with other DNA satellites associated withbegomoviruses, contain nucleotide stretches of begomoviral origin,presumably the remains of recombination events involved in theirorigin.

Results and discussion

Identification of a small circular DNA molecule in Malvastrumcoromandelianum infected with Sida golden yellow veinvirus-Malvastrum

In a recent survey of begomoviruses infecting weeds in Cuba,we identified a new strain of the bipartite begomovirus Sida goldenyellow vein virus (SiGYVV-Ma) infecting a plant (sample 177) ofthe malvaceous species Malvastrum coromandelianum (Fiallo-Olivéet al., 2012). Digestion with HindIII and NheI of DNA amplified byrolling circle amplification (RCA) from sample 177 rendered a highamount of linear product of ~700 bp, in contrast with the loweramount of begomoviral genome linearized with the appropriateenzymes (Fig. 2). We cloned the ~700 bp DNA fragments obtainedafter digestion with HindIII or NheI and obtained the complete se-quence of five clones (two for HindIII and three for NheI). The clonedsequences were 687–689 nt in length (See Table 1 for GenBank ac-cession numbers) and had a nucleotide identity of 99.3–100% be-tween them. Based on comparison with known DNA viruses andsatellites, these molecules appeared to be circular single-strandedDNA and contained several features of geminivirus origin, includinga stem–loop with the conserved nonanucleotide TAATATTAC, aTATA box about 160 nt upstream from the stem–loop resemblingthe Rep gene promoter region, and iteron-like motifs located just up-stream from the TATA box (Fig. 3A, C, D). The iteron-like sequencesare associated, as shown for ToLCV-sat (Dry et al., 1997; Lin et al.,2003), with a stem–loop structure (Supplemental Fig. 1). They alsocontain an A-rich region, with three peaks of high A content, a hall-mark of betasatellites and alphasatellites (Briddon et al., 2003,2004) (Fig. 3B). In addition to the characteristics mentioned above,a nucleotide database search revealed a stretch of 23 nt (AGCCT-TAGCTTCGCCGGAGCTGA) located between the TATA-box and thenonanucleotide that showed 95.7% identity with a region containedin the SCR of numerous betasatellites (Fig. 3C). Analysis of the codingregions indicated the presence of four small ORFs (38–53 aa) with noobvious RNA polymerase II promoter elements required to initiate

A B

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dIII

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aII

Nh

eI

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este

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eI

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eI

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eI

228 404 412 413 414 424

*

* *

Fig. 2. (A) Agarose gel electrophoresis of RCA-amplified DNA from Malvastrum coromandelianum sample 177 digested with EcoRI, HindIII, HpaII, NheI and SacI. (B) Agarose gel elec-trophoresis of RCA-amplified DNA from S. micranthum sample 404 and M. coromandelianum samples 228, 412, 413, 414 and 424 digested with HindIII, HpaII and NheI. Arrowheadsindicate the undigested DNA. Arrows indicate the linearized full length begomoviral components. Asterisks correspond to the putative DNA satellite molecules.

3E. Fiallo-Olivé et al. / Virology 426 (2012) 1–6

transcription, suggesting that, as shown for ToCLCV-sat, these mole-cules may rely on geminiviral and/or host factors for replicationand systemic spread (Dry et al., 1997). The Rep binding-like regioncontained three iteron-like copies with a GGGG core identical tothat present in SiGYVV-Ma DNA-A. One of the iteron-like copies isinverted and has no counterpart in the DNA-A (Fig. 3D). This stronglysuggests that these molecules are associated with SiGYVV-Ma anduse the viral Rep for replication. In other words, we have foundwhat seems to be a novel type of DNA satellites associated withNew World bipartite begomovirus infections.

A novel class of begomovirus-associated DNA satellites widely distributedacross Cuba

In an attempt to identify additional DNA molecules similar tothose found in sample 177, we analyzed symptomatic samples of

able 1NA satellite molecules isolated from M. coromandelianum and S. micranthum plants.lone designation, size and GenBank accession numbers are indicated.

Plant host Sample Clone Size (nt) GenBank accession no.

M. coromandelianum 177 H1 687 JN986808H3 689 JN819486N2 687 JN819487N4 687 JN819488N7 687 JN819489

228 H1 677 JN819490H3 677 JN819491H5 677 JN819492H6 677 JN819493H6B 677 JN819494

412 N1 686 JN819498N2 687 JN819506N3 686 JN819499

413 N1 684 JN819507N2 684 JN819500N3 684 JN819501N5 684 JN819502

414 N1 686 JN819503N2 686 JN819504N3 686 JN819505

424 N1 683 JN819508N2 683 JN819509N3 683 JN819510

S. micranthum 404 N1 685 JN819495N2 685 JN819496N3 685 JN819497

TDC

the malvaceous M. coromandelianum, Sidastrum micranthum and anunidentified species. Forty out of 69 samples (32 of M. coromandelia-num, 6 of S. micranthum and 2 of the unidentified species) analyzedby PCR for the presence of begomovirus DNA-A (Rojas et al., 1993)tested positive (Supplemental Table 1). The sequencing of ampliconsand comparison with sequences deposited in GenBank demonstratedthat all corresponded to partial sequences of DNA-A components ofspecies belonging to the Abutilon clade of New World begomoviruses(Rojas et al., 2005) (Supplemental Table 1).

In six of the begomovirus-infected samples, amplification by RCAand digestion with several restriction enzymes rendered a largeamount of DNA with a predicted molecular weight slightly lowerthan 750 bp. The sample numbers were 228 (digested with HindIII),404, 412, 413, 414 and 424 (digested with HpaII and NheI) (Fig. 2).In the same samples, the amount of begomovirus DNA observedafter amplification by RCA and digestion, when present, was muchlower than that observed for the ~750 bp molecules (Fig. 2). TheDNA molecules from sample 228 (digested with HindIII) and samples404, 412, 414, and 424 (digested with NheI) were cloned and se-quenced. These molecules were 677 to 686 nt in length (see Table 1for GenBank accession numbers) and the nucleotide identity betweenclones derived from a single sample ranged from 98.5% to 100%,whereas nucleotide identity between samples ranged from 71.8% to97.5% (Supplemental Table 2).

All sequenced molecules presented the unique features observedin the molecules isolated from sample 177, i.e., a stem–loop withthe conserved nonanucleotide TAATATTAC, a TATA box and iteron-like sequences (associated with a stem–loop structure) characteristicof begomoviruses (Fig. 3D), an A-rich region (Fig. 3B), and a short re-gion with high nucleotide identity with the SCR of betasatellites(Fig. 3C). The iterons (three copies) of the DNA satellites differedfrom those (two copies) of the DNA-A of the begomoviruses presentin the same samples (Fig. 3D). It is worth mentioning that the mole-cules cloned from the only sample from S. micranthum that was ana-lyzed (sample 404) contained an imperfect TATA-box (TCTA), andonly two G were conserved in the core of the three iteron-like se-quences present within them (Fig. 3D). As for the sequence obtainedfrom sample 177, the apparent chimeric nature of these moleculescould be the result of recombination events involved in their genera-tion, which could involve at least a begomovirus DNA-A and a betasa-tellite. The somewhat blurred TATA box and iteron-like motifspresent in the S. micranthum sample could represent a further stepin the evolution of these satellites.

Recently, a vector-enabled metagenomic (VEM) approach con-ducted in southern Florida showed the presence of circular DNA

stem-loop stemstem--looploop

nonanucleotide

betasatellite-related sequence

C

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llite

s

TATA Iteron-like sequences D

B

Ade

nine

con

tent

(%

)

I II

III

200 600 400

Nucleotide position

0

25

50

75

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177H1

A

Fig. 3. (A) Schematic representation of the DNA satellite molecule (clone 177H1) isolated fromM. coromandelianum (sample 177). The colored areas correspond to the A-rich region(green), the iteron-like region including the TATA box motif (blue), the betasatellite-related sequence (red), and the conserved stem–loop (yellow). (B) Adenine percentage alongthe sequence of the DNA satellite molecule (clone 177H1), indicating the three A-rich peaks (I, II, III). (C) Alignment of the betasatellite-related (in red) and conserved stem–loopregion (in yellow) of the consensus sequences for the DNA satellites molecules isolated from each plant sample (indicated in the left). The homologous region present in the sat-ellite conserved region from selected betasatellites are shown below. *Mimosa yellow leaf curl betasatellite (DQ641710), #Tomato yellow leaf curl Thailand betasatellite (GQ994097)and ~500 more betasatellite sequences, @Chili leaf curl betasatellite (JF706231) and ~40 more betasatellite sequences. (D) Alignment of the iteron-like sequences and TATA box mo-tifs of the DNA satellites characterized in this work (a consensus sequence is given for each plant sample) with the iteron region present in the DNA-A of the begomoviruses presentin each plant sample. The strictly conserved nucleotides in the iterons are highlighted in blue.

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molecules in adults of the whitefly B. tabaci, a natural vector of bego-moviruses, which were tentatively named whitefly VEM satellites(WfVEM-Sat) (Ng et al., 2011). In the latter study, the genome struc-ture of such molecules was not described in detail nor were the pre-sumed helper virus(es) determined, since the whitefly viromescontained a diverse range of begomoviral sequences from both bipar-tite New World begomoviruses and the monopartite Old World To-mato yellow leaf curl virus. The fact that these molecules wereputatively present in virions in the whiteflies, had an A-rich region,and did not code for a replication-associated protein, suggested thatthey were DNA satellites associated with begomoviruses. Our phylo-genetic analysis demonstrated that these molecules are closely relat-ed to some of the DNA satellites that we found in Cuba, and are

included in one of the clusters that we identified (see below). Thefinding that the DNA satellites found in M. coromandelianum and S.micranthum are associated with infections of New World begomo-viruses belonging to the Abutilon clade support that these constitutea novel class of DNA satellites, which also includes the WfVEM-Satmolecules. Phylogenetic analysis showed that DNA satellites foundin M. coromandelianum form two main clusters, corresponding tosamples from western and eastern Cuba, respectively (Fig. 4). Themolecules from the only S. micranthum sample clustered with, butwas distinct from, the M. coromandelianum molecules from easternCuba. Thus, it seems that both geographic and host factors determinethe distribution of genetic variability of the DNA satellite moleculesdescribed in this work. Interestingly, WfVEM-Sat molecules are

M. coromandelianum (western Cuba)

M. coromandelianum (eastern Cuba)

S. micranthum

WfVEM-Sat-h WfVEM-Sat-d WfVEM-Sat-f WfVEM-Sat-c WfVEM-Sat-e WfVEM-Sat-a WfVEM-Sat-b WfVEM-Sat-g 424N3 424N1 424N2 413N5 413N1 413N2 413N3 414N3 414N2 414N1 412N1 412N3 412N2

228H3 228H1 228H6B 228H6 228H5 177H3 177N2 177H1 177N7 177N4 404N2 404N3 404N1

ToLCV-sat

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Fig. 4. Phylogenetic tree illustrating the relationships among the novel satellite mole-cules described in this work and those obtained from B. tabaci in Florida [WfVEM-Satfrom Ng et al. (2011), GenBank accession numbers HM859904–HM859911]. The treewas constructed using the Neighbor-Joining method with MEGA 4.1. Branchessupported with less than 60% bootstrap values (1000 replicates) were collapsed. Theevolutionary distances were computed using the p-distance method. The ToLCV-satsequence (GenBank accession number U74627) was used as outgroup.

5E. Fiallo-Olivé et al. / Virology 426 (2012) 1–6

more closely related to the DNA satellites from western Cuba, whichis close to Florida.

We predict that RCA-based analysis will reveal the presence ofDNA satellite molecules related to those described here in otherbegomovirus-infected plant species in the Caribbean and other re-gions in the NewWorld. The observation that DNA satellite moleculesfound in S. micranthum are distinct from those found in M. coroman-delianum suggests that the complexity of this novel class of DNA sat-ellites may be higher than we have shown here. Furthermore, recent

424

404412 413 414

Fig. 5.Map of Cuba showing the location of weed sampling (white circles), with indication ofmovirus infection in each province.

metagenomic studies have shown the presence of previously unchar-acterized circular ssDNA satellites in an Antarctic lake (López-Buenoet al., 2009), thus opening the possibility that novel satellites associ-ated with ssDNA viruses may be found in new environments.

In addition, the sequence features shown by the novel type ofsatellites reported here, reminiscent of those found in ToLCV-sat,raise a question regarding how are they replicated by the helpervirus(es). Some of the iteron-like sequences associated with the sec-ondary stem–loop structure could be involved in trans-replication,as shown for ToLCV-sat (Dry et al., 1997; Lin et al., 2003). ToLCV-sat and betasatellites have been shown to be replicated by a numberof helper viruses (Dry et al., 1997; Nawaz-ul-Rehman et al., 2009;Saunders et al., 2008). Partial sequencing of DNA-A of begomovirusespresent in the same plant samples in which the novel satellites werefound showed a level of genetic variability compatible with a highlevel of replication promiscuity. Future work with infectious clonesshould clarify this point, and ascertain whether the presence ofthese DNA satellites influences the accumulation of begomovirusgenome components or whether they play a role in pathogenicity,as demonstrated for betasatellites and some alphasatellites.

Materials and methods

Sample collection, DNA extraction and rolling circle amplification

Symptomatic Malvastrum coromandelianum, Sidastrum micranthumand unidentified malvaceous plants were collected from differentregions across Cuba (Fig. 5 and Supplemental Table 1). All plantsexhibited different degrees of yellow mosaic. Total DNA was isolatedfrom leaf samples as previously described (Crespi et al., 1991). Circu-lar DNA was amplified by rolling circle amplification (RCA) with φ29DNA polymerase using the TempliPhi DNA Amplification Kit (GEHealthcare, USA) (Inoue-Nagata et al., 2004) and RCA products weredigested with several restriction enzymes. DNA fragments with asize that corresponded neither to begomoviral genome componentsnor to fragments derived from them after digestion were cloned inpBluescript II SK(+).

Begomovirus detection by polymerase chain reaction

To detect begomoviral DNA-A, universal primers designed byRojas et al. (1993) with slight modifications were used: MA55:GCCCACATYGTCTTYCCNGT (based on PAL1v1978) and MA56:GGCTTYCTRTACATRGG (based on PAR1c476).

177

228

samples containing DNA satellite molecules (figures within circles) associated to bego-

6 E. Fiallo-Olivé et al. / Virology 426 (2012) 1–6

Sequence analysis

EditSeq and SeqMan [available in MEGALIGN (DNAStar Inc.,Madison, WI, USA)] were used to analyze and assemble the sequences.BLAST (http://www.ncbi.nlm.nih.gov/) was used for sequence similar-ity searches. Multiple sequence alignments and distance calculationwas conducted using CLUSTAL V (Higgins et al., 1992) for DNA-A se-quences or CLUSTAL W (Thompson et al., 1994) for the novel satellitemolecules (also included in MEGALIGN). MEGA 4.1 (Tamura et al.,2007) was used for phylogenetic inference by the Neighbor-Joiningmethod. The search for A-rich regions was carried out using the onlineDNA base composition analysis tool (http://molbiol-tools.ca/Jie_Zheng/dna.html).

Supplementary materials related to this article can be foundonline at doi:10.1016/j.virol.2012.01.024.

Acknowledgments

The authors are grateful to Rob W. Briddon for critical reading ofthe manuscript. The work in Cuba was partially supported by theInternational Foundation for Science, Stockholm, Sweden, through agrant (C/4778-1) to E. F.-O. The work in Spain was partially carriedout in the frame of the network 111RT0433 from Fundación CYTED.J. N.-C. and E. M. are members of the Research Group AGR-214,partially funded by the Consejería de Economía, Innovación y Ciencia,Junta de Andalucía, Spain (cofinanced by FEDER-FSE). E. F.-O. wasrecipient of an MAEC-AECID fellowship (Ministerio de AsuntosExteriores y de Cooperación, Spain).

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