viruses Article A Virus in American Blackcurrant (Ribes americanum) with Distinct Genome Features Reshapes Classification in the Tymovirales Thanuja Thekke-Veetil 1, Thien Ho 1, Joseph D. Postman 2, Robert R. Martin 3 ID and Ioannis E. Tzanetakis 1,* ID 1 Department of Plant Pathology, Division of Agriculture, University of Arkansas System, Fayetteville, AR 72701, USA; [email protected] (T.T.-V.); [email protected] (T.H.) 2 National Clonal Germplasm Repository, United States Department of Agriculture, Corvallis, OR 97333, USA; [email protected] 3 Horticultural Crops Research Unit, United States Department of Agriculture, Corvallis, OR 97331, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-479-575-3180; Fax: +1-479-575-7601 Received: 21 April 2018; Accepted: 26 July 2018; Published: 3 August 2018 Abstract: A novel virus with distinct genome features was discovered by high throughput sequencing in a symptomatic blackcurrant plant. The virus, tentatively named Ribes americanum virus A (RAVA), has distinct genome organization and molecular features bridging genera in the order Tymovirales. The genome consists of 7106 nucleotides excluding the poly(A) tail. Five open reading frames were identified, with the first encoding a putative viral replicase with methyl transferase (MTR), AlkB, helicase, and RNA dependent RNA polymerase (RdRp) domains. The genome organization downstream of the replicase resembles that of members of the order Tymovirales with an unconventional triple gene block (TGB) movement protein arrangement with none of the other four putative proteins exhibiting significant homology to viral proteins. Phylogenetic analysis using replicase conserved motifs loosely placed RAVA within the Betaflexiviridae. Data strongly suggest that RAVA is a novel virus that should be classified as a species in a new genus in the Betaflexiviridae or a new family within the order Tymovirales. Keywords: Betaflexiviridae; blackcurrant; Ribes americanum virus A; characterization; detection 1. Introduction Technological developments in high throughput sequencing (HTS) have resulted in the rapid discovery and characterization of several novel plant viruses [1–4]. The USDA National Clonal Germplasm Repository (NCGR) maintains and distributes various accessions of specialty fruit and nut crop species from around the world [5]. This includes currant (Ribes spp.), a berry known for its potential health effects [6]. This study was initiated when an American blackcurrant maintained in the USDA-NCGR, Corvallis, Oregon (Ribes americanum; PI 617879) showed virus-like symptoms (Figure1). The plant was infected with two viruses [ 3]: A waikavirus [7] and an undescribed virus in the order Tymovirales. Viruses 2018, 10, 406; doi:10.3390/v10080406 www.mdpi.com/journal/viruses Viruses 2018, 10, 406 2 of 10 Viruses 2018, 10, x FOR PEER REVIEW 2 of 10 Figure 1. Symptoms observed on American blackcurrant infected with Ribes americanum virus A. Figure 1. Symptoms observed on American blackcurrant infected with Ribes americanum virus A. Infected Ribes americanum cultivar Gall (PI 617879, left) showing ragged leaf margins and crinkled leaf Infected Ribes americanum cultivar Gall (PI 617879, left) showing ragged leaf margins and crinkled leaf surface compared to healthy plant (right). surface compared to healthy plant (right). The order Tymovirales includes five families, namely Alphaflexiviridae, Betaflexiviridae, GammaflexiviridaeThe order Tymovirales, Deltaflexiviridaeincludes, and Tymoviridae five families, [8]. Members namely Alphaflexiviridaeof this group are, non-enveloped,Betaflexiviridae, Gammaflexiviridaeflexuous, filamentous, Deltaflexiviridae viruses primarily, and Tymoviridae infecting plants,[8]. Members and have of genomes this group resembling are non-enveloped, eukaryotic flexuous,mRNAs [9]. filamentous This manuscript viruses primarilydescribes infectingthe charac plants,terization and of have a novel genomes virus resembling tentatively eukaryotic named as mRNAsRibes americanum [9]. This manuscript virus A describes(RAVA). theThe characterization evolutionary relationship of a novel virus of RAVA tentatively with named other as known Ribes americanummembers of virusthe order A (RAVA). Tymovirales The evolutionary indicated relationshipthat RAVA ofis RAVAa virus with with other unique known genome members features, of the orderpossiblyTymovirales representingindicated a new that genus RAVA in the is a Betaflexiviridae virus with unique family, genome or being features, the type possibly representative representing of a ayet new to be genus described in the family.Betaflexiviridae Discoveryfamily, of this or novel being virus the type adds representative information to of the a yetcurrent to be knowledge described family.on this Discoverydiverse group of this of novelviruses, virus and adds expands information the number to the of current virus species knowledge reported on this in diversethe order. group of viruses, and expands the number of virus species reported in the order. 2. Materials and Methods 2. Materials and Methods Double-stranded RNAs were extracted from 20 g of symptomatic leaves from currant accession PI 617879Double-stranded (Figure 1) and RNAs subjected were extractedto a degenerate from 20oligonucleotide-primed g of symptomatic leaves reverse from transcription-PCR currant accession PI 617879 (Figure1) and subjected to a degenerate oligonucleotide-primed reverse transcription-PCR (DOP RT-PCR) followed by HTS and sequence assembly as described [10]. The FirstChoice® RLM- ® (DOPRACE RT-PCR)Kit (ThermoFisher followed Scientific, by HTS andWaltham, sequence MA, assembly USA) was as used described to obtain [10 the]. 5 The′ terminus. FirstChoice The 5′ RLM-RACE Kit (ThermoFisher Scientific, Waltham, MA, USA) was used to obtain the 50 terminus. and 30′ RACE-RT-PCRs0 were performed using virus-specific primers and programs described in Table TheS1. For 5 and RACE 3 RACE-RT-PCRs and confirmation were of performedgenome region usings virus-specificdownstream of primers ORF 1 and total programs nucleic acids described were inextracted Table S1. as For described RACE and [3]. confirmationFor all amplification of genome reactions, regions downstreamTakara LA Taq of ORFpolymerase 1 total nucleic (Takara acids Bio wereUSA extractedInc, Mountain as described View, CA, [3 ].USA) For allwas amplification used according reactions, to the manufacturer’s Takara LA Taq instructions. polymerase All (Takara PCR Bioproducts USA Inc,were Mountain sequenced View, for CA,at least USA) three-fold was used coverage according of to the the regions. manufacturer’s The complete instructions. nucleotide All PCRsequence products of RAVA were sequencedwas deposited for at in least GenBank three-fold under coverage accession of thenumber regions. MF166685 The complete. nucleotide sequenceThe genome of RAVA organization, was deposited putative in GenBank ORFs, under and protein accession sequences number were MF166685. derived using the NCBI ORFThe Finder genome [11]. organization,RNA binding putativeamino acid ORFs, (aa) and resi proteindues present sequences in RAVA were derivedproteins usingwere theidentified NCBI ORFusing Finder Pprint [ 11(Prediction]. RNA binding of Protein amino RNA-Interact acid (aa) residuesion; [12]) present and transmembrane in RAVA proteins domains were identifiedidentified usingusing PprintTMHMM (Prediction Server v. of 2.0 Protein [13]. The RNA-Interaction; secondary structures [12]) and of transmembranethe proteins were domains predicted identified by PSI- usingPred, TMHMMI-TASSER Server [14,15], v. 2.0and [13 nucl]. Theear secondary localization structures signals of identi the proteinsfied with were cNLS predicted Mapper by PSI-Pred,[16]. The I-TASSERprotein sequences [14,15], andof members nuclear belong localizationing to signalsthe families identified in the withorder cNLS Tymovirales Mapper were [16]. obtained The protein from sequencesGenBank of(Table members 1) and belonging aligned towith the the families virus in puta the ordertive proteinsTymovirales usingwere Clus obtainedtalW on from Bioedit GenBank [17]. (TablePhylogenetic1) and aligned analyses with were the performed virus putative using proteins the maximum using ClustalW likelihood on method Bioedit with [ 17]. 1000 Phylogenetic bootstrap analysesreplicates were on MEGA performed v. 7 [18] using and the phylogenetic maximum likelihoodtrees displayed method using with Treeview 1000 bootstrap in MEGA replicates v. 7. on MEGA v. 7 [18] and phylogenetic trees displayed using Treeview in MEGA v. 7. Viruses 2018, 10, 406 3 of 10 Table 1. Details of virus isolates used for the phylogenetic studies of Ribes americanum A. RdRp-RNA dependent RNA polymerase, MTR-Methyl transferase, α-Alphaflexiviridae, β-Betaflexiviridae, γ-Gammaflexiviridae, L-Deltaflexiviridae, T-Tymoviridae. Virus Acronyms Virus Names Genus (Family) RdRp/MTR ACLSV Apple chlorotic leaf spot virus Trichovirus (β) NP040551.1 CMLV Cherry mottle leaf virus Trichovirus (β) AOY07780.1 GVA Grapevine virus A Vitivirus (β) AFV73358.1 GVB Grapevine virus B Vitivirus (β) AIL90366.1 CNRMV Cherry necrotic rusty mottle virus Robigovirus (β) NP059937.1 AOPRV African oil palm ringspot virus Robigovirus (β) YP002776347.1 CtChV-1 Carrot Ch virus 1 Chordovirus (β) AHA85534.1 CtChV-2 Carrot Ch virus 2 Chordovirus (β) AHA85531.1 DiVA Diuris virus A Divavirus (β) YP006905850.1 DiVB Diuris virus B Divavirus (β) AFV57240.1 AVCaV Apricot vein clearing associated virus Prunevirus (β) AKN09002.1 CPrV Caucasus prunus