Accepted Article 1 [email protected] [email protected] contact details: Other E mail address 2502 +6189360 Phone: 2424+ 6189360 Fax: author *Corresponding Australia Biotechnology Centre,School of Veterinary and Life Sciences, Murdoch University, Perth, W.A. 6150, Japan 305-8566, Ibaraki, Tsukuba, Higashi, 1-1-1 6, Central Tsukuba (AIST) Technology and Science Industrial Advanced of Institute National 2 M. Saqib characterisation. new a of identification Serendipitous Article :Original Type Article This article This protectedis by Allcopyright. rights reserved. 10.1111/ppa.12293 doi: lead to differences betweenversionthis and the Versionof Record. Please cite this article as through thebeen typesetting,copyediting, pa This article hasbeen accepted for publicationand undergone fullpeer review but has not Plant Section, Plant Biotechnology Research Group, Western Australian State Agricultural Agricultural State Australian Western Group, Research Biotechnology Plant Section, Virus Plant Institute. Research Bioproduction Group, Research Regulation Gene Plant *1,2 . , S. J. Wylie J. , S. : [email protected] 2 , M. G. , K.Jones M. Iflavirus 2 .

Title Page -like virus infecting tomato, and its subsequent subsequent its and tomato, infecting virus -like gination and process, which proofreading may Accepted Article

RNA-dependent RNA(RdRp). polymerase Pairwise analysis of the deduced amino acid of replicase viral approximately 207 kDa with co coat protei intothree subsequently processed 8,506of nucleotides. It to is predicted encode a 314.5kDa, which single polyprotein of is plants ( tomato sequence genomic The apreviouslyof virus undescribed was identified symptomless from Abstract abbreviation: New Keywords title Running [email protected] This article This protectedis by Allcopyright. rights reserved. the of members resembling virus of plant-infecting report a first virus)genus Matilda within thefamily (Tomato created Tomavirus be the that propose and further, virus name (TMaV), matilda Tomato Weproposethe virus. by RT-PCR of the viral replicative strand, and sequence of the revealed RdRp itshares that identityclosest the withmembers of family : -like, insect virus, plant virus virus plant virus, insect picornavirus-like, : , genus : Tomato matilda virus matilda : Tomato Solanum lycopersicum tomato matilda virus (TMaV). (TMaV). virus matilda tomato Iflavirus Iflavirus (19-47% identity). Evidence of replication in plants wasof replication plants detected Evidence in identity). (19-47%

from tomato ). The viral genome is a positive sensea positive ssRNA is molecule genome The viral ). n components of 13.7, 17.9 and 13.5 kDa, and akDa, 13.7, and of 13.5 and 17.9 n components

short interfering RNAs the matching (siRNAs) nserved a motifs for helicase, a protease, and Iflaviridae Iflaviridae . . This the This is . Accepted Article clearly recognizable as that of a virus. Aspects of the putative genome sequence of the virus, its virus, its of the sequence genome putative of the Aspects virus. a that of as recognizable clearly polyprotein a encodes (ORF) frame reading open thelarge , described with identity sequence Presencevirus-like a of wassequence revealed assembly after contigs. of Despite very low levels of platform. theIllumina using analysed were (siRNAs) RNAs and short-interfering mRNA total study, from aseedling a for transcriptomeretailer study on responses to phytoplasma infection. In this obtained originally plants in tomato virus anovel of discovery theserendipitous wepresent Here discovered from apparently healthy specimens (Coetzee (Coetzee specimens healthy apparently from discovered infectionwild in plants isnot surprising, but even healthy, virus-free plants, has often revealed an abundance of viruses in such plants (e.g. Wylie Wylie (e.g. plants such in viruses of abundance an revealed often has plants, virus-free healthy, apparently from RNA analyse to technologies sequencing of deep application Recent viruses. ‘symptomless’ or ‘latent’ as designated are symptoms visible not induce do that Viruses hosts. infection were evident, viewhas and this most lead that the virusesto induce symptoms in their important economically on studies previous most In sequences. genomes of complete dept the and needed), is sequence or structure virus the of knowledge previous no (i.e. generic are approaches thatNGS-based is technologies (Kreuze viruses plant new of thediscovery to applied been have deep sequencing, and (NGS), sequencing generation as variously known technologies sequencing New Introduction This article This protectedis by Allcopyright. rights reserved. al ., 2012; Roossinck, Wylie etal 2013 2012; et al ., 2009; Roossinck 2012; Wylie a,b ). Perhaps the incidence of asymptomatic virus h of sequencing sometime enables the determination determination the enables sometime sequencing of h in well-studied plant species new viruses are being being are viruses new species plant well-studied in high throughput nucleotide sequencing, next- sequencing, nucleotide throughput high et al virus infections of plants, visible symptoms of symptoms visible plants, of infections virus ., 2012). The ., The major2012). advance overearlier et al ., 2010). ., 2010). et et Accepted Article benthamiana, N. tabacum tabacum N. benthamiana, sativum Pisum RNeasy Plant Miniprep kit (Qiagen) following manufacturer’s the instructions. After grinding 100mg tomato leaf material in nitrogen,liquid total was RNA extracted using an extraction RNA seedlingsma wasto used sativum vulgaris capsicum tomato, of each plants five of leaves onto rub-inoculated and (Aquacel) earth sodium 0.05M containing pH 7.2 buffer phosphate ( to tomato ( light. cultureswere Virus transferred natural under 20-24°C at glasshouse aninsect-proof in mix potting standard a using maintained were Tomato wereseedlings obtained from in 2009 seedling a Perth, supplier in Western Australia. They Plant materials maintenanceand virus methods and Materials classificationwithin the family is described hosts alternative to transmission This article This protectedis by Allcopyright. rights reserved. Capsicum annuum Capsicum (coriander). Five control plants of each species were mock inoculated. Grafting to tomato tomato Grafting to inoculated. mock were species each of plants control Five (coriander). (common bean) (common (pea) ). For sap inoculation, infected leaves were ground in 0.05M potassium potassium 0.05M in ground were leaves infected inoculation, sap For ). , Solanum tuberosum tuberosum , Solanum , Vicia faba , Vicia intain virus intain virus inoculum. (tobacco) Iflaviridae (broad bean) (broad , Chenopdium quinoa, Daucus carota Daucus quinoa, , Chenopdium . (potato) here. We discuss implications of its proposed proposed its of implications discuss We here. Solanum lycopersicum) , Raphanus sativus , Raphanus , Solanum melongena , Solanum sulphite. The sap was mixed with diatomaceous with mixed The diatomaceous was sap sulphite. (radish) , capsicum and chilli plants plants chilli and capsicum , (egg plant) (carrot) and (carrot) , Brassicarapa , Nicotiana , Nicotiana Choriandrum Choriandrum , Phaseolus , Phaseolus (canola) , Accepted Article free water and RNA extracted from uninfected plants. plants. uninfected from extracted RNA and water free 95°C for 3min and 40 cycles of 95°C for 15sec and 60°C sec. for 45 Negative controls were RNase- by min followed 15 for 42°C were conditions Cycling 1). (Table probe of nM and250 primers reverse was performed in triplicate. Each reaction contained 50 mM mM MgCl 50 contained reaction Each triplicate. in performed was perf was realtime step PCR One min. 10 for °C 72 of extension afinal and min for 1 72°C at extension pair), primer each for (calculated sec 30 °Cfor 56 48°C- at Incubationconditions were 94°C for 5min,cyclesof 25 denaturation 94°C10 sec, for at annealing mM KCl,pH 8.0) 150 contained 1/20 diluted cDNA as template, 2.5 mM MgCl mM 2.5 template, as cDNA diluted 1/20 contained (25µl) mixtures Reaction (Fermentas). Kit Synthesis cDNA Strand First H Minus a RevertAid™ using synthesis cDNA strand first for used were primers hexamer random or primers reverse Specific Reverse Transcriptionand PCR The primer pairs were designedconsensus from regions 500-800 nt apart. analysis. sequence genome for whole version 1.82 Program Alignment Sequence Multiple ClustalW with aligned were sequences TMaV full-length Two 1). (Table by PCR amplified was cDNA the cDNA A tosynthesise primer 1). was designed (Table DNA Te Integrated using designed were probe, TMA real pair internal time with primer TMA PCR,reverse forward together andTMA quantitative the For assays. PCR RT diagnostic confirm to and sequencing, dideoxy Sanger using it resequence the primers were (Table 1) across TMaV toof designed genomeA series overlapping andused amplification PCR for primers TMaV of Design This article This protectedis by Allcopyright. rights reserved. μ M dNTPs, and 10 ormed using Jumpstart Ready Master Mix (Sigma). reaction (Sigma). Each ReadyMasterMix using Jumpstart ormed μ M of each primer and 0.5 unit unit and0.5 primer each of M from the viral replicative (negative) strand, and strand, (negative) replicative viral fromthe chnologies (Coralville, Iowa) online software software online Iowa) (Coralville, chnologies 2 , 1 X reaction buffer (10 mM Tris-HCl, 50 50 Tris-HCl, mM (10 buffer reaction X , 1 2 , 300

Taq Taq nM each of forward and DNA polymerase. Accepted Article the cDNA using an Illumina GAIIx platform (Illumina Inc. (Illumina platform GAIIx Illumina an using cDNA the Macrogen Inc (Seoul, South Korea) forcDNA librar to sent was plants three from collected tissue leaf from ethanol) in µgsuspended (20 RNA Total Sequencing Generation Next sequence. TMaV Illumina-generated original sets. primer adjacent by amplified sequences complementary the from data comparing by ends or 5’ 3’ the at were confirmed Sequences 1.0.3. SeqEd and assembled (Hall 1999) were using BioEdit products PCR sequencing the After with an Applied Biosystems sequencer. 3730 Sequencingwas repeated twice for bothDNA strands. Dye Big using done was sequencing Sanger primers. weresequenced set primer each from products PCR products were purified from agarosegel using aQIAquickGel Extraction Kit (Qiagen). Amplified sequencing Sanger This article This protectedis by Allcopyright. rights reserved. GeneiousPro v5.0.4(Drummond D synthesis. cDNA primed randomly and ntfragments 18-30 for size fractionation after analysed was siRNA samples,was mRNA sequenced cDNAafter synthesisusing oligo-dT primers.the For other sample, to interrogate the GenBank database (NCBI) using nt (Blastn) and deduced amino acid (aa) (aa) acid amino and deduced (Blastn) nt using (NCBI) database GenBank the to interrogate (Invitro package NTI Vector the in included is which Contigs were produced and further assembled into Katrinebjerg, Denmark) and Velvet 0.6.04 (Zerbinov using & Birney 2008) default parameters. e novo sequence assembly of the 78 nt reads was done using three short-read assemblers: assemblers: threeshort-read using done was reads nt 78 of the assembly sequence et al., 2010), CLC Genomics Workbench (CLC bio Finlandsgade Finlandsgade bio (CLC Workbench CLC Genomics 2010), Sanger-generated sequences were mapped to the to mapped were sequences Sanger-generated directly after purification using 5’ and 3’ end end 3’ and 5’ using purification after directly y synthesis of mRNA and single end sequencing of of endsequencing single and mRNA of y synthesis gen, Carlsbad, CA). Assembled contigs were used wereused contigs Assembled CA). Carlsbad, gen, longer contigs using the program ContigExpress, ContigExpress, program the using contigs longer Terminator 3.1(Perkin-Elmer,Terminator Foster City, CA),

San cycles.San Diego, CA)over78 For two RNA Accepted Article sequences. (http://www.ebi.ac.uk/Tools/pfa/iprscan), and by identity after alignmentwith characterised virus theNCBI v6.0, Pro Geneious by predicted were proteins deduced of identities of ORFs, Positions annotation Genome sequences. virus ICTV to and genomics ITAG2.4 fu wereanalysed sequences unmatched remaining recorded 100were analysis.than Blastscores lower Group (ITAG) database, and the data was downloaded from SolGenomic Networks for further Annotation Tomato International the at sequences against (NCBI) MegaBlast using analysed were sequences translatedall from six frames entire (Blastx). Theassembly of 23,271contig sequences This article This protectedis by Allcopyright. rights reserved. 1965). Pauling, & model (Zuckerkandi correction Poisson the using computed were identity joining, Theaa scores methods. maximum minimum parsimony evolution and Theev sequences. between persite substitutions residue in aa length branch tree phylogenetic the toinfer used distances of evolutionary those as units same the lengths branch with scale, to drawn were Trees 2). (Table included also were diversityviruses of the studied were selected for phylogenetic analysis.Picorna-like viruses of plants the of members of sequences available Publically viruses. related other of 21 those with together TMaV of sequences aa (Tamura MEGA5 within implemented model Cedergren (Thompson W Clustal using Alignment analysis Phylogenetic Iflaviridae et al., representing the genetic, host range, and geographical geographical and range, host genetic, the representing 1994) and phylogenetic analysis using a Sankoff-Morel- a using analysis and phylogenetic 1994) Conserved Domain Database (CDD), InterProScan InterProScan (CDD), Database Domain Conserved , mature peptides, and domains encoded by them bythem encoded domains and ,peptides, mature olutionary history was inferred using neighbor- using inferred was history olutionary rther using Blastn to GenBank tomato mRNA, tomato mRNA, GenBank to Blastn using rther as no significant hit ag significant no as et al., 2011) was done on deduced replicase replicase deduced on done was 2011) ainst the database. The thedatabase. ainst Accepted Article code HQ260868. accession GenBank granted was TMaV Aof Isolate is proposed. (TMaV) virus matilda Tomato name the which for virus, undescribed previously a of those as designated were sequences these analyses, sequence After this. confirm to done not was (RACE) ends cDNA of amplification rapid 5’ although region of (UTR) 104nt and3’ UTR ofnt, 113 su untranslated 5’ the and polyadenylated, were sequences genome virus The signals. poly-adenylation total) 3’ excluding of reads, assembled 0.01% B 2 3,570 from nt (isolate plant from and8,506 total) of 0.04% reads, 13,325 from assembled 1 plant A from (nt)(isolate nucleotides 8,506 of contigs were and 32,359,875 reads of 78nt. Contigswere assembled 31,558,959 generated tested tomato plants two from isolated mRNAs total of sequencing Illumina analysis sequence and assembly Genome Results This article This protectedis by Allcopyright. rights reserved. provided in supplement data S3. are results Genbank complete The VDV-1. with matches closely that sequence virus new is the 576) virus-1 destructor are which sequences, viral match sequence contig Three S3. data supplemental in provided are results Genbank and file raw data complete The viruses. matched sequences contig Three S2). of 96.6 total A supplementaldata. provide in S1 was granted accession the no SRX506967. entire The assembled contig sequences 23,271 are and (NCBI) Archive Read Short the to submitted was one sample from sequences ofIllumina set The (VDV-1), (VDV-1), Grapevine leafroll-associatedvirus ggested thatvirus the was sequence complete, % of the contigs matched tomato sequences (Fig (Fig sequences tomato matched contigs the % of de novo. novo. de and and The longest virus-like sequences virus-likesequences longest The Lone Star virus Star Lone . One contig. One (no Varroa Varroa Accepted Article primer pairs, and their sequences determined. Th theirsequences determined. primer pairs, and ofexpe the 1).Genome fragments primer pairs(Table entireTM ampliconsto generated were the amplify RT-PCR overlapping sequencing, Illumina by generated sequence genome of the accuracy confirm To Resequencing infection. geno virus the mapto that RNAs small of presence 6,278 where there was 8-foldcoverage, and nt 7,396-7,416, which had 12fold coverage. The where there was 40-fold coverage, at nt 4,228-4,250 where was there 14foldcoverage, at 6,256- nt 1a). wereThese the 5’at nt UTR 61-83where there was 2,197-fold coverage, at nt 2,534-2,556 (Fig. genome TMaV the of regions five only to mapped sequences RNA Thesmall sequence. genome ntlong 20-26 of sequences 24,010,322 sequenced. was with tomatoplant infected TMaV A third This article This protectedis by Allcopyright. rights reserved. sequence. genomic virus the identity to 100% with mapped compliment reverse and it plant and negativecontrol (water) did not amplify a product. The amplicon of 481 wasnt sequenced, TMaV-inf five in strand negative the detected 1) infected from detected was virus ofRNA strand the evidenceFurther of virus replication inpl tomato shown). qPCR RT 1)for (Table TMAprobe together with the pair primer reverse TMA and forward TMA The studies. range host in TMaV detect to subsequently wereused 1) (Table F2/R2 pairs Matilda primer The obtained. sequence original the with identity

and small RNA species generated were then then were generated species RNA and small ected tomato plants while RNA from the uninfected from while RNA the ected plants tomato ants was provided when the replicative (negative) (negative) thereplicative when provided was ants e re-sequenced genome shared 99.8 % sequence %sequence 99.8 shared genome re-sequenced e me provides evidence of a host response to TMaV TMaV to response host a of provides evidence me were obtained. These were mapped to the TMaV TMaV the to mapped were These obtained. were was also used for TMaV diagnostics (data not not (data diagnostics TMaV for used also was plants. Specific primers TMaV RCF, RCR1 (Table (Table RCR1 RCF, TMaV primers Specific plants. aV genome by the Sanger method using specific specific using method theSanger by aV genome cted sizes (667-836 nt) were obtained from all Accepted Article twoweeks post-inoculation. Only infected and shorter than described iflaviruses, whose genomes range in size from 8.7 kb SBV to 10.9 kb. The kb. to10.9 kb SBV 8.7 from size in range genomes whose iflaviruses, described than shorter is this tail: poly(A) the excluding length, ntin 8,506 was TMaV of A isolate genomeof RNA The characterisation Molecular microscope. whenvisualized bylight theleaves on were observed insects No asymptomatic. remained plants systemically-infected other the whereas plants, of two of plants Only plants. tomato TMaV-infected from sap with inoculated carota D. quinoa, C. tabacum, N. benthamiana, N. tuberosum, of each plants five TMaV, of range host and development symptom about more understand to order In Virus inoculation toalternate hosts This article This protectedis by Allcopyright. rights reserved. iflaviruses SB (AAAAAUGGC), not the but and (GAAAAUGAG) flexiviruses, the including viruses, plant some of motifs initiation translation The first in-frame AUGoccurred 105-107at nt withinthe context CAAAAUGGA,which resembles genome ispredicted toencode a single polyprotein from asingle was There ORF. a of UTR 104nt. 5’ (rhv-like) structural proteins with sequence identity identity sequence with proteins structural (rhv-like) the most similar architecture were iflaviruses, including small SBVand Three LLV. rhinovirus-like th viruses other with homology on based predicted, followed a3’ by UTR of 113nt and apoly-A tail.domains Active within the polyprotein (Fig. 1a) were calculated mass of 314kDa. It terminatedis by an (isolate LLV-1) (CACUAUGGC). Thepredicted polyprotein is2,787 aa residues in length with a S. lycopersicum, C. annuum C. lycopersicum, S. S. melongena S. were positive for TMaV in RT-PCR and qPCR analyses on new, uninoculated leaves leaves uninoculated new, on analyses qPCR and RT-PCR in forTMaV positive were Apple stem grooving virus grooving stem Apple , S. melongena, S. V (isolate Korea) (UAUUAUGGA) V (isolate and Korea) C. annuum annuum C.

(AAAAAUGAG), and the iflavirus, VDV-1 VDV-1 theiflavirus, (AAAAAUGAG), and P. vulgaris P. ochre (UAA) terminationcodon 8,466-8,468,at nt at share similar domain architecture. Those with with Those architecture. domain similar share at to iflavirus capsid proteins were identified at the plants developed a mild mosaic on the leaves theleaves on mosaic mild a developed plants , V. faba, R. sativus, B. rapa, P. sativum, S. S. sativum, P. rapa, B. sativus, R. faba, V. and and C. sativum sativum C. S. lycopersicum, C. annuum C. lycopersicum, S. Hardenbergia virus A virus Hardenbergia were manually manually were Lygus lineolarisvirus-1

,

Accepted Article and G, aa residues 2,228 and 2,231, respectively. A definitive VPg region was not found butin TMaV SBPV (ADI46683), DWVand (ADK55525). Nucleotide binding sites were at C-terminal the present H were identified by homologywith insectpicornaviruses including SBV(NP_049374), LLV (AEL30247), aa residue 2,076, at the E 2,115, andcatalytic the within C the motif GxCG (aa residues 2,209-2,212) catalytic Hlocated 3Csuperfamily. protease at of protease, The cysteine the members a cleaved by domain is located residues 2,061-2,235.at Picornav residues 1,376-1,383 and Bmotifthe asVILVD residues at 1,425-1,429. A cysteine protease-like (Walker residue hydrophobic a his where (hhhh[D/E]), motif B theWalker and residue, any xis where motif (GxxxxGK[S/T]) A Walker the by characterised are which superfamily, domain NTPase P-loop with the homology shares helicase (Ehrenfeld capsids arenon-enveloped polyproteins their of terminus theN- at proteins capsid similar three encode that Viruses 1b). (Fig. kDa 13.4 of mass a has 977 of 13.7 kDa, rhv-like 2at residues 571-728 with amass of 17.9 kDa, and rhv-like 3atresidues 858- prot 1 Rhv-like polyprotein. the of N-terminus This article This protectedis by Allcopyright. rights reserved. of regions to VPg identity ofsequence areas residuesat 2,515-2,528,(MDYSNFGPCFH) residuesand2,543-2,553. IV at 2,463-2,469, II (VFCASPVDYTIALRQNLLHFCAATMKN) at residues 2,482-2,508, III (AVGINPLGPEWSKI) residues 2,607-2,659. Other conserved RdRp domainscharacterised at I were residues (TLKDERR) replicase core motif Tx3S/TGx3 NS/Tx22 GDD (Koonin was 1991) present as SGx3 GDD Tx3 NSx39 at conserved The residues. acid amino conserved of number a containing motifs several by formed The RdRp was identifiedaa at residues 2,316-2,782. catalyticThe centre virus of RNA RdRps is ) at 2,024-2,048 (TxxxxxYxxxxxxxxxxxxFRxQxV) were at2,024-2,048 (TxxxxxYxxxxxxxxxxxxFRxQxV) ) ) at 1,359-1,379) at (DxxQxxYxxVP) and et al., et 2009).An helicaseRNA predicted is from residues 1,374-1,480. TMaV The et al., Cowpea mosaic Cowpea ein was located at aa residues 307-427 and has mass a andhas residues307-427 aa locatedwas at ein Tomato ringspot virus ringspot Tomato 1982). In the TMaV A motif exists as GASGIGKS at plus-strand ssRNA animal viruses with icosahedral icosahedral with viruses animal ssRNA plus-strand iral proteins are expressed as a single polyprotein polyprotein a single as expressed are proteins iral identified that may represent a VPg. aVPg. may represent identified that (CPMV) (Family (CPMV) (Family Secoviridae , genus , genus , genus , genus Accepted Article

name of this new monospecific genus be ‘Tomavirus’ ( ‘Tomavirus’ be genus monospecific new this of name coverage 97-100%, e-values 4 (40-52%, identities maximum shared also rhv1-3 proteins structural individual The 2). (Fig. Korea 93-99%, e-values 3 confirmed TMaV’sclose association with iflaviruses. Iflaviridae Tomato we that propose similar architecture, genome therelatively close Because se of 28%) identities with of TMaV. that virus wilt bean Broad 2 - 0.0 e-values coverage, (85-95% identity aa 31-39% of maximum a shared TMaV of sequence polyprotein entire The analysis Phylogenetic This article This protectedis by Allcopyright. rights reserved. plant. a in replicating to arthropod an in replicating from journey improbable virus’s ancestral the to refers name the Thus, places. foreign to mantravelling toanitinerant refers that 1895 in Paterson Banjo by written Matilda’ ‘Waltzing ballad Australian theiconic from chosen sequences of virusa forwhich we propose the name Tomato matilda virus. Thename matildawas from extracted RNA of analysis Deep sequencing Discussion the within genus asecond that propose we arthropods, ( Eptesicus fuscus Eptesicus . Due to the fact it replicates in plants, unlike unlike plants, in replicates it thefact Dueto . ) (Table 3). RdRP proteins of plant-infecting secoviruses secoviruses plant-infecting of proteins RdRP 3). (Table ) -108 , CPMV, –2 -154 -108 ) with SBV, LLV-1, and an unclassified picorna-like virus that infects bats ) with those of SBV isolates from Korea, China and UK, and LLV-1 from from LLV-1 and UK, and China Korea, from isolates SBV of those ) with Rice tungro spherical virus spherical tungro Rice -22 –3 quence identity of the TMaV genome and gene products, and the and products, gene and genome TMaV the of identity quence -52 ) with isolates of SBV and LLV-1. Analysis of the RdRp domain domain the RdRp of Analysis LLV-1. and SBV of with isolates ) asymptomatic tomato plants revealed the genome thegenome revealed plants tomato asymptomatic It maximum shared (34-41%, It identities coverage matilda virus be classified within the family family withinclassified the be virus matilda To other described iflaviruses that all replicate in in replicate all that iflaviruses other described , and , and Iflaviridae mato Tomato torrado virus torrado Tomato ma tilda be created. We propose the that propose We created. be virus Apple latent spherical virus spherical latent Apple ). ). shared lower (20- lower shared ,

Accepted Article is suffici presented theevidence we feel together, Taken response. defensive a mounting was host thatthe indicating sequence, viral genome by supported was This hosts. plant in replication of evidence also is plants other to transmission and byRT-PCR sequence theviral of strand systemic infection evidenceis strong that replicatesTMaV in plants.Detection of the replicative subsequent and plants between Transmission plants. solanaceous these between transmitted asymptomatically and induced mild symptoms of infection in some capsicum plants. It was virus. plant a is TMaV that evidence strong as sequences arthropod-derived no were There output. geneti its arthropod, an from derived were sequence reads raw from constructed contigs thatmost showed results analysis Blast sample. the contaminated had virus that an arthropod represented thesequence that apossibility was there discovered, was of TMaV genome the When This article This protectedis by Allcopyright. rights reserved. Bunyaviridae infecting genus (Blackberry picornavirus virus of the Z) sister family member proposed plant-infecting a of Recent identification plant. a in replicate wasto able step molecular of theidentity notably plant, to a questions arealso There plants. other in pathology st particle range, host natural its of transmission, mode its establishing include These answered. be to remain virus unusual this about questions Many (Ullman (Ullman Tospovirus et al ., 2005),., are evidence that viruses have thismade transition before. within the predominantly arthropo predominantly within the were from either the tomato plant or from TMaV. If the virus Ifthe TMaV. from or tomato plant the either were from ent to ascribeTMaV as a plant virus. Furthermore, TMaV infected tomato and eggplant eggplant and tomato infected TMaV Furthermore, s needed before the probable insect virus ancestor ancestor virus insect probable the before s needed the detection of small RNAs that mapped to the tothe thatmapped RNAs small of detection the ructure, replication cycle in different hosts, and and hosts, different in cycle replication ructure, about its probable transition from an arthropod arthropod an from transition probable its about , c signature would be clearly evident in the Blast inthe evident cclearly would be signature identified from over 23,000 contigs. This stands stands This contigs. over 23,000 from identified d and vertebrate infecting family family infecting vertebrate d and and the existence of plant- the of existence the and Accepted Article containing a superfamily III helicase, a 3C-lik a helicase, III superfamily a containing module three-domain a (iv) and particles, non-enveloped icosahedral, small form which domains proteins capsid (iii) polyprotein(s), processed encoding a polyprotein, hence the name (Christian & Scotti 1998; Hunter Hunter 1998; Scotti & (Christian name hence the polyprotein, a encoding genome, family membersof the members. infecting arthropod of consists the of member unclassified an as proposed is Z virus blackberry Secoviridae al., Secoviridae polymerase. thefamiliesorder includes The 5 a with order the to belongs it that suggests found virus the of sequence genome the of Analysis This article This protectedis by Allcopyright. rights reserved. Oers, Current 2010). virus paralysis bee (DWV), species of have date to that particles icosahedral enveloped, non- are members, otherunclassified the and (ICTV), Viruses of Taxonomy onthe Committee International by the described species iflavirus seven All thegenome. of end the5’ at located Members of genusthe Hashimoto 2009). 2010). 2010). Plant infect Picornavirales Ectropis obliqua virus obliqua Ectropis ’ -bound VPg and 3 and VPg -bound Iflavirus (including the sub-family sub-family the (including and . Like TMaV, members of the order exibit exibit order members of the LikeTMaV, . Iflaviridae, : (SBPV) (van Oers 2010). To date, (van Oers 2010). (SBPV) iflavirusesseven accepted are ICTV (vanby the is Dicistroviridae Dicistroviridae Iflaviridae Infectious flacherie virus virus flacherie Infectious ing members of the ing members Iflavirus ’ and there are four unassigned members (Le Gall unassigned members (Le four are and there -polyadenylation signal, (ii) genome translation into auto-proteolytically auto-proteolytically into translation genome (ii) signal, -polyadenylation Dicistroviridae (EoV), (EoV), family members infect insects from the orders Lepidoptera, Lepidoptera, orders the from insects infect members family within the within and Perina nuda virus nuda Perina Comovirinae Iflaviridae. Iflaviridae.

Two new insect infecting families have been reported in Picornavirales Iflaviridae are characterised by the presence of two cistrons each each cistrons two of bypresence the characterised are e cysteine proteinase, and an RNA-dependent RNA RNA anRNA-dependent and proteinase, cysteine e Picornaviridae organized in a module containing three related related three containing module a in organized (IFV), and other species are are species other and (IFV), Although bothmonopartite families have a ) (Sanfacon ) (Sanfacon only been reported to infect arthropods. The type type The arthropods. infect to reported been only have a single ORF and structural genes are are genes structural and ORF have asingle (PnV), (i) a positive-sense RNA genome,positive-sense RNA (i) a usually within are predominantly the family , Dicistroviridae Sacbrood virus Sacbrood et al., Dicistroviridae 2009), 2009), but theplant infecting et al., Deformed wing virus wing Deformed , (SBV), VDV-1 and VDV-1and (SBV), et al et , which otherwise 2006; Valles & ., 2008; Kapoor Kapoor 2008; ., , Slow Slow

et et Accepted Article throughput methods of biological characterisation wi characterisation of methods biological throughput high new that envisage We behind. further lag will viruses of new characterisation biological methodical of approach classical the that probable it is and rapidly, beidentified will taxa virus New ones. new of creation the force and groupings taxonomic current challenge will these and viruses, rapidThe developmentdeep sequencing of technologies will identify greater numbers of unusual further. bestudied to needs concept This insects/animals. to TMaV of transmission the for ‘vector’ a as acting fact in is host plant the that possibility also a is there plants in replicate to ability its and TMaV of organisation genome on the Based 1988). (Bradbear, andapiculture 1964) Kuruta, & (Aizawa production silk in losses economic substantial for responsible as identified have been They kb. Orthopte and Diptera, Heteroptera, Hymenoptera, This article This protectedis by Allcopyright. rights reserved. to conflict of interest no declare. have authors from Mitsuda Nobutaka Dr thank We fellowship. JSPS and Development and Research of Division University and Murdoch (CaPRI) Research Science wasMS supportedcontributions by from the Murdoch University Institute for Crop and Plant Acknowledgements years. coming in community science the challenge inevitably will that life of domains all from discoveries new virus AIST for providing bioinformatics support. All All support. bioinformatics providing for AIST ra, and have a linear ssRNA(+) genome of 8.8-9.7 8.8-9.7 of genome ssRNA(+) alinear and have ra, ll be developed to cope to bedeveloped ll with the ‘tsunami’ of of the ‘tsunami’ with Accepted Article L.). L.). Stoltz DB,Genetic 2010. characterization paralysis of slow bee virus honeybee (Apis of the mellifera G, Berth Cordoni B, Locke B, Dainat JR, de Miranda General Virology General virus. beeparalysis with acute comparison and virus bee Kashmir of sequence nucleotide Complete 2004. SM, Camazine DB, Stoltz CE, M, Cameron Shen S, Tyler M, Drebot JR, de Miranda Metagenomic analysis of the viromes of three North American bat species: Viral diversity diversity Viral species: bat American North three of viromes the of analysis Metagenomic Donaldson EF, Haskew AN, GatesJE,Huynh J, Moore CJ,Frieman MB, 2010. analysis viruses infecting grapevines:ofof vineyard. virome a Coetzee B, Freeborough MareeHJ, MJ, Celton JM, Rees DJG, JT, Burger Deep 2010. sequencing (eds). Plenum, New Pp York. 301–336. 1998. PD, Scotti PD, Christian pests. and diseases honeybee major of distribution World 1988. N, Bradbear silkworm,the cond under aseptic Infection 1964. KurutaK, Aizawa K, References This article This protectedis by Allcopyright. rights reserved. 1549. virusof thehoney bee:an insectpicorna-like virus. RC,Ghosh Ball BV, Willcocks MM, CarterMJ, 1999. The nucleotide sequenceof sacbrood Virology virusidentifiedpicorna-like in the T,Fujiyuki TakeuchiH, M,Ono S, Ohka Sasaki T, Nomoto A, Kubo T, 2004. Novel insect Vaccines Ehrenfeld E, Modlin J, Chumakov K, 2009. Future of polio vaccines. 2010a. A, T, Wilson, Thierer S, Stones-Havas R, Moir M, Kearse J, Heled M, Cheung B, Ashton AJ, Drummond 13018. among different batspecies thatshare a commonhabitat. Journal of General Virology General of Journal Geneious

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Accepted Article nucleotide binding fold. fold. binding nucleotide kinases myosin, synthase, ofATP beta-subunits Walker JE, Saraste M, RunswickGay MJ, NJ, Distantly 1982. related sequencesthe alpha- in and 361. positive-strand RNA virus infecting the red imported fire ant, ant, fire redimported the infecting virus positive-strand RNA Valles SM, Hashimoto Y,2009. Isolationcharacterization and of Solenopsis invicta virus 3,a new pp. 231–250. Norfolk, Press, Academic Virology Caister. van Oers, M.M., 2010. Genomics and biologyof Iflaviru 4931–4932. determinants of insect transmission. Ullman DE, WhitfieldGerman AE, TL, 2005.Thrips and tospovirusescome mappingof age: 555–561. fleck virus polyprotein: evidence of affinities with . picornaviruses. with affinities of evidence polyprotein: virus fleck yellow parsnip of the analysis 1993. Sequence AF, ReavyB, Turnbull-Ross MA, Murant Mayo AD, weight matrixchoice. and penalties gap position-specific weighting, sequence through alignment sequence multiple progressive of sensitivity the improving W: CLUSTAL 1994. TJ, Gibson DG, Higgins JD, Thompson methods. parsimony maximum and distance, evolutionary likelihood, maximum using analysis genetics evolutionary Molecular MEGA5: 2011. S, Kumar M, Nei G, Stecher N, Peterson D, Peterson K, Tamura ofriceorganization tungro spherical virus. Shen P,Kaniewska M,SmithC, Beachy RN, 1993.Nucleotide sequenceand genomic This article This protectedis by Allcopyright. rights reserved. pooledin cultivatedand wild plantsamples. WylieSJ, LuoH, HuaJones Li, 2012. MGK,Multiple polyadenylated RNA virusesdetected 20 by isregulated RNA virus paralysis Wilson JE, PowellMJ, HooverSarnow P,SE, , 4990–4999. Molecular Biology and Evolution Evolution and Biology Molecular Nucleic Acids ResearchNucleic Acids EMBO Journal EMBO Proceedings of the National Academy of Sciences (USA) Sciences of Academy National the of Proceedings

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74 , , Accepted Article bold. in indicated are virus matilda Tomato of that with identity overall highest with genes order the of members confirmed 24 of polymerase Table 3 press:York New andLondon. 97-166. In: Evolving Genes and Protein PaulingZuckerkandi E, 1965. L, divergence Evolutionary convergenceand in proteins.Pp. graphs, Zerbino DR, Birney E,2008. Velvet: algorithms for de novo short read assembly using Bruijn de General Virology ofmember the Picornaviridae associatedwith acutegastroenteritis inhumans. S, 1998. Complete nucleotide sequence and genetic organization of Aichi virus, a distinct T,Yamashita Sakae K, Tsuzuki H,Y,Suzuki Ishikawa N,TakedaN, MiyamuraT, Yamazaki inAustralia. species) wildinfect populations and captive collections of temperate terrestrial orchids ( WylieSJ, LiH, Dixon KW,RichardsH, JonesMGK, 2013 DOI:10.1371/journal.pone.0079587 from Australian terrestrial orchids. WylieSJ, LiH, Jones MGK, 2013 This article This protectedis by Allcopyright. rights reserved. Table 2 replicative strand negative Table 1 legends Table Pairwise comparisons of amino acid sequence identity of the RNA-dependent RNA RNA RNA-dependent the of identity sequence acid amino of comparisons Pairwise Members of the order order the of Members Primer sequencesused for detection, geno Gene Research Research Gene

72 , 8408–8412. 18 Virus Research , 821–829. of tomato matilda virus. of tomato virus. matilda Picornavirales

a . Donkey Orchid Symptomless Virus: A Viral ‘Platypus’ PLoS ONE

171 . Edited by V. Bryson and H. J. Vogel. Academic Academic Vogel. J. H. and Bryson V. by Edited :22-32. used for phylogenetic analysis. analysis. phylogenetic used for 8(11): e79587. me amplification and identification of the the of identification and amplification me Picornavirales Picornavirales b . Exotic indigenousand viruses and Tomato matilda virus. The The virus. matilda Tomato and Journal ofJournal Diuris

Accepted Article Table 1 reported. is host the su acid amino in distance evolutionary an indicates at Numbers parameters. software thedefault with MEGA5 using conducted was analysis thephylogenetic and ClustalX using werealigned polymerase order members of the Fig 2 polyprotein. the in positions their and (blue), proteins regulatory and structural for coding and domains showing scale) to (drawn genome viral Schematic toscale). positionsto (drawn (For genome the mapped RNAs small where regions the indicate line gray the on redsquares The anddomains. genes Fig 1 Figure legends This article This protectedis by Allcopyright. rights reserved. identification negative of (replicati the Neighbor-joining tree of the deduced amino acid sequences of twenty-four confirmed confirmed twenty-four of sequences acid amino deduced of the tree Neighbor-joining Predicted (a) organisationgenome of Tomato ma 1038 357- 1-829 Matilda F age Cover 1821 1004- 2277 1745- Primer sequences used for detection, ampl detection, for used sequences Primer name Primer Matilda F0 Matilda F1 aid 2 TTATTGTCGA 2 CTTTACGCACTGCTTCCTT R2 GTATAACTTTTGGTCCGGAA Matilda F2 Picornavirales Forward Reverse Reverse ACAACG ATGATATTATATGTTATAAT Forward AGGCGC AGGCGC ATCACGACAGAGAGGACAA A ACGGCAGCCACGCTAAGAA and tomatomatilda virus. Amino acid sequences for the entire ve) strand of tomato matilda virus. virus. tomato of matilda ve) strand of additional motifs in the RdRp see text). text). (b) see RdRp the in motifs of additional open reading positions frame(green), genes of nodes indicate bootstrap scores. The scale bar bar scale The scores. bootstrap indicate nodes bstitutions per site. For each virus, the of order the site. virus, For each per bstitutions ification of overlapping genome fragments, and and fragments, genome overlapping of ification tildaviruslocations showingproposed the of R0 R R1 CCACG GACGACATAATGCATTTT CAACTT TATTCCGCTAAAACTTTTC T GG TCATTATGGCGCCTGTAT Size Size Amplicon 681 829 532 817 Accepted Article This article This protectedis by Allcopyright. rights reserved. 3006 2256- 3684 2967- 4371 3655- 5064 4345- 7809 7195- 7214 6485- 6511 5755- 5788 5015- 7349 7829- Replicative pair strandprimer 8506 7785- 7975 8062 7890- QPCR for used Primers Matilda F3 Matilda F4 Matilda F5 Matilda F6 F10 Matilda R9 TTTACCTTGTGCTGTTGCAG F9 Matilda R8 CAGCCCGACGTGATTATCAT Matilda F8 Matilda R7 GTGGTTGCTGCTTAAGAATG F7 F11 Matilda probe probe TMA forward TMA RCF RCF TMaV A AAAAAGGAAGCAGTGCGTA CCTG GTTGCTAATTTTTATGGCATT G GAGTGGTGTTAGATCCAAA G TTAATCTAGTTACTGATGCC AATTAACAACGTCTGCAGGT AATTAACAACGTCTGCAGGT CAAGGTACAGTTTACCAGCA CAAGGTACAGTTTACCAGCA FAM5’ATGACTTGATCGCCGCTGTCTCAGAA3’BHQ1 FAM5’ATGACTTGATCGCCGCTGTCTCAGAA3’BHQ1 GCGCC TCTTTAGCCCAAGAACATT C GGCTCCTGATGGGCTGCCA

1 RCR v Re R3 R4 R5 R6 0 R1 1 R1 GA GA CGTCCCATTCTATAGCAG T TCCGCTTCTTCTTTGGATC CT TGTATGCGGCATCAGTAA CCAGTGTCTGCAACAGCA TA GGAATTGAGTTTCGATGA CT CACCAAACACAAGACCAA TT ACCTGCAGACGTTGTTAA CA TA TA TCTGTTGCTGGTAAACTG AGCGAT AGCGAT ACGATACCATAACGTGCC CAAAT TTCTATCTACACTATCATA GTGCC GTGCC CCGGAGGAAGGGTGTT 750 481 717 716 756 773 719 729 614 172 720 Accepted Article

Table 2 This article This protectedis by Allcopyright. rights reserved. virus paralysis bee Slow virusdwarf virus disease Foot-and-mouth virus paralysis Aichi virus not assigned yet number accession *Protein VDV YP Netherlands ToRSV 145791 Australia TMaV HQ260868* SBPV YP_003622540 UK SBV SBV SBV UK RTSV PYFV NC003628 USA USA PLV ADR79389 MCDV NC003628 USA LLV AEL30247 KKV YP Japan KBV 015696 FMD South USA DHV DQ812092 Korea UK DWV AAP49283 DWV ADK55525 DCV CpMV CrPV BBWV BBPV YP_001285409UK ALSV AV Virus GenBank

AZ82 Korea ADZ98922 NP China UK ADN38255 049374 Members of the order order the of Members (AV); (MCDV); (CrPV); NP_620765.1 AAA66056.1 NP_851403 AJ539141 NP_044945 NP_613283.1 NP_647481 AAD38152 BAA90870 NC_001918 NC_001918 accession Apple latent spherical Parsnip yellow fleck virus virus fleck yellow Parsnip Cowpea mosaic virus (SBPV); (FMD); Tomato matilda virus Canada USA USA UK Australia USA USA Korea South Japan Japan origin Geographical Kashmir bee virus

virus Picornavirales (CpMV); (ALSV); . (PYFV); Brevicoryne brassicae picorna-like virus picorna-like brassicae Brevicoryne Drosophila C virus C Drosophila Picorna-like virus virus Picorna-like (TMaV); (TMaV); lycopersicum lycopersicum Bos taurus Bos Vigna unguiculata annuum Capsicum brassicae Brevicoryne aradestructor Varroa idaeus Rubus S cerana A. sativa Oryza Pastinaca sativa fuscus Eptesicus A. mellifera mellifera A. mellifera A. A. mellifera mellifera A. melongaster Drosophila commodus Teleogryllus domestica Malus A. mellifera mellifera A. platyrhynchos Anas mellifera A. A Source Common name sapiens Homo Host name A. mellifera mellifera A. Zea mays lineolaris Lygus (KBV); olanum olanum p is mellifera used for phylogenetic analysis. analysis. phylogenetic used for Kakugo virus Kakugo Tomato ringspot virus (DCV); (PLV); (KKV); Deformed wing virus Rice tungro spherical virus virus tungro spherical Rice Raspberry Raspberry ReddickMaize bug plant Tarnished Tomato This study This study Tomato Cattle Mason Cowpea Unpublished Capsicum Ryabov 2007 Cabbage aphid ieOngus Mite Unpublished honeyAsiatic bee Shen Rice Donaldson Turnbull-Ross Parsnip bat brown Big oe e Ghosh Ma Honey bee Honey bee Honey bee Moore Moore bee Honey Honey bee Fruit fly Wilson Cricket Apple Li oe e Fujiyuki Honey bee Lanzi Duck Kim bee Honey Man Yamashita Honey bee Lygus lineolaris virus lineolaris Lygus (ToRSV); (BBPV); Varroa destructor virus destructor Varroa (DWV); virus wilt Broadbean (LLV); (RTSV); de Miranda Miranda de 1998 Johnson and Christian 1983 Shanks and Lomonossoff de Miranda Miranda de Rott Unpublished Duck hepatitis virus hepatitis virus Duck et al Maize chlorotic chlorotic Maize et al et et al et et a et et al et al et Sac brood virus brood Sac ., 2000 et a et al et a et et al et al et al ., 2011 et al et l ., 2007 ., 1995 ., 1993 1993 ., ., 2006 2006 ., (VDV). l et al et l et al ., 2004 ., 1999 ., 2011 ., 2003 ., 2000 et al et al ., 2004 ., 1997 et al (BBWV); ., 1998 ., 2010 ., 2004 2004 ., ., 2010 2010 ., ., 1993 (DHV); (SBV); Cricket

(USA) (USA) virus paralysis Cricket (USA) virus bee Kashmir (USA) virus spherical tungro Rice virus (USA) (USA) virus dwarf chlorotic Maize virus (UK) (UK) virus fleck yellow Parsnip (Japan) virus latent spherical Apple (USA) (USA) virus mosaic Cowpea (USA (USA Picorna-like virus (Australia) virus matilda Tomato (USA) virus lineolaris Lygus (Australia) C virus Drosophila Korea) Korea) virus(South Sacbrood Korea) (South wiltvirus bean Broad virus (UK) (UK) virus Slow bee paralysis 24. virus(UK) Sacbrood (China) virus Sacbrood rvcrn rsia 2.222222223 3 31 2 2 2 2 2 2 2 2 Accepted 23. brassicae Brevicoryne Article 2. inTableprovided withtheare RdRp TomatomatildaIdentities virus indicated bold.in Accession codes ofpolymerase 24members oftheorder Table 3 This article This protectedis by Allcopyright. rights reserved. Pairwise comparisons ofdeduced amino acid sequences ofthe RNA-dependent RNA 5 24. 4 23. 8 28. 5 26. 6 29. 5 39. 6 24. 6 20. 8 24. 9 23. 9 31. 9 24. a nad Ca V RS To 1 24. 9 6 24. 2 3. 2 5 3. 2 1 6. 2 3 6. 2 8 6. 2 5 2. 3 4 6. 4 5 3. 2 7 5. 2 8 2. 2 7 2. 2 4 5. 2 ea or K h ut o S V W B B 7 5. 2 7 5. 2 2 5. 2 8 4. 2 5 3. 2 0 3 4 0. 3 6 0. 3 2 2. 3 4 5. 2 2 4. 2 7 2. 2 8 4. 2 9 4. 2 A S U V M p C 5 3. 2 2 5. 2 9 4. 2

7 7. 2 4 2 7 1. 3 2 2. 3 4 3

6 6. 2 9 2 5 2 8 5. 2 3 7. 2 n a p Ja V S L A 6 6. 2 1 7. 2 3 7. 2

3 4. 2 1 7. 2 5 8. 3 5 4. 3

4 6. 2 5 8. 2 9 5. 2 8 5. 2 K U V F Y P 8 6. 2 7 5. 2 4 6. 2 8 6. 2

9 5. 2 9 4. 2 3 1. 6

9 7. 2 2 4. 2 1 6. 2 5 3. 2 2 7. 2 A S U V D C M 8 5. 2 5 7. 2 2 7. 2 7 6. 2 7 2 6 4. 2 9 8. 2 3 6. 2 3 7. 2 A S U V S T R 3 6. 2 7 2 3 6. 2 1 6. 2 8 3 9 2 7 . 4 2 2 . 9 3 5 . 5 . 4 2 A S U V B K 6 . 6 2 7 . 6 2 1 . 6 2 1 . 7 2 7 . 7 2 1 3 2 . 4 2 2 . 6 6 A S U V P r C 6 . 8 2 1 . 4 2 6 . 8 2 6 . 8 2 Picornavirales .3 29 .9 31 .6 24 a ali str Au V C D .4 28 .8 25 .1 28 .4 28 8 3 4 . 0 4 A S U V L L 9 . 3 4 3 . 7 2 9 . 3 4 2 . 4 4 a ali str Au V Ma T .4 39 47 32 .7 28 .3 46 47

A S U V L P 1 . 6 4 333 33 3 23 9 . 0 3 1 . 6 4 3 . 6 4 ea or K V B S

2 0. 3 4 7. 9 9 7. 9 with that of Tomato matilda virus.with of that Tomato matilda a n hi C V B S 4 0. 3 4 7. 9 K U V B S 4 . 0 3

K U V P B S

K U V BP B L N V D V A S U V W D K U V W D n a p Ja V K K ea or K V H D

an Jap V A K U D M F (USA) (USA) Deformed wing virus auovrs(aa) 20. virus(Japan) Kakugo disease virus (UK) (UK) virus disease Foot-and-mouth 18. (Japan) virus Aichi (UK) (UK) virus picorna-like (South Korea) Korea) (South hepatitis virus Duck virus-1 (Netherlands) (Netherlands) virus-1 destructor Varroa Accepted Article (UK) Deformed wing virus

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Accepted Article

This article This protectedis by Allcopyright. rights reserved.