Arch Virol (2011) 156:1665–1669 DOI 10.1007/s00705-011-1015-y BRIEF REPORT Blackberry virus E: an unusual flexivirus Sead Sabanadzovic • Nina Abou Ghanem-Sabanadzovic • Ioannis E. Tzanetakis Received: 5 November 2010 / Accepted: 30 April 2011 / Published online: 9 June 2011 Ó Springer-Verlag 2011 Abstract A virus, named blackberry virus E (BVE), was disease (BYVD), a serious disorder observed in the south- recently discovered in blackberries and characterized. The ern United States [19]. Disease symptoms are not specific to virus genome is 7,718 nt long, excluding the poly-A tail, any given virus combination, and different virus combina- contains five open reading frames (ORFs) and resembles that tions are found associated with identical symptoms [19]. of flexiviruses. Phylogenetic analysis revealed relationships This study was initiated with four blackberry plants to allexiviruses, which are known to infect plants of the showing BYVD-like symptoms observed in northeastern family Alliaceae. BVE lacks the 3’end-proximal ORF, Mississippi (Fig. 1A). They were tested by ELISA using which encodes a nucleotide-binding protein, a putative antibodies specific for 12 viruses and a ‘‘universal potyvirus’’ silencing suppressor in allexiviruses. The overall results of kit (Agdia Inc., USA). Additionally, they were tested by this study suggest that this virus is an atypical and as yet reverse transcription polymerase chain reaction (RT-PCR) undescribed flexivirus that is closely related to allexiviruses. using specific primers for viruses identified recently in BYVD-affected plants, or one still being characterized and Keywords Blackberry Á Virus Á dsRNA Á lacking serological diagnostics (authors, unpublished data). Alphaflexiviridae Á RT-PCR All four specimens were infected with blackberry virus Y, a virus known to be asymptomatic in single infections [17], leading to the assumption that one or more additional viruses Rubus species (blackberry, raspberry and their hybrids) are are involved in the observed symptomatology. Double- hosts to more than 40 viruses [12], with several new species stranded RNAs (dsRNAs) were isolated as described [20] identified in the last few years. Most of the new viruses have and were further purified by selective digestions with DNase, been found in association with blackberry yellow vein RNase and proteinase K. Uniform patterns of dsRNAs with multiple high-molecular-weight bands were observed in extracts from all specimens, strongly suggesting the pres- Sequence data were deposited in GenBank as accession number ence of the same or closely related virus(es) (Fig. 1B). JN053266. This pattern was reminiscent of flexivirus infections (the term ‘‘flexivirus’’ applies generally to members of the Electronic supplementary material The online version of this article (doi:10.1007/s00705-011-1015-y) contains supplementary families Alpha-, Beta- and Gammaflexiviridae), and material, which is available to authorized users. therefore, ‘‘universal’’ degenerate primers for these viruses [4] were applied in order to generate initial nucleotide & S. Sabanadzovic ( ) Á N. Abou Ghanem-Sabanadzovic sequence data. RT-PCR using dsRNA as a template yielded Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, amplicons of 366 bp from all samples (not shown). PCR Mississippi State, MS 39762, USA products from different samples showed 94-100% nucleo- e-mail: [email protected] tide identity (97-100% aa identity), confirming infection by the same virus. BLASTx searches showed that the virus I. E. Tzanetakis Division of Agriculture, Department of Plant Pathology, shares high aa identity with members of the family University of Arkansas, Fayetteville, AR 72701, USA Alphaflexiviridae. 123 1666 S. Sabanadzovic et al. Fig. 1 A. Line patterns and vein yellowing/feathering symptoms observed on original BVE sources. B. Double- stranded RNAs extracted from BVE-infected blackberries (lanes 2 and 3) compared to dsRNAs of Cryphonectria hypovirus 1 (lane 1) and peanut stunt virus (lane 4). Note the pattern similarity extracted from two different blackberry specimens (lanes 2 and 3) The sequenced region had 78-83% identity to the cor- ORF 1 encodes a 1499-aa protein with a molecular mass responding genome portion of several members of the of 168.6 kDa (p169). BLASTp analysis of this protein genus Allexivirus. This close similarity led to the devel- identified conserved Tymovirales motifs for a methyl- opment of degenerate primers for members of this genus, transferase (pfam 10641), helicase 1 superfamily (pfam which were used to obtain additional sequence information 01443) and RNA-dependent RNA polymerase II super- for this virus (a list of degenerate primers is provided in family (pfam 0978). The closest orthologs of the polypro- Table S1). Degenerate primers were successfully applied tein are encoded by allexiviruses (garlic virus A, GarV-A; in RT-PCR to amplify portions of the flexivirus present in garlic virus C, GarV-C; garlic virus E, GarV-E; garlic virus diseased blackberries. Genome sequence gaps were X, GarV-X; and shalot virus X, ShVX), with 60%-62% obtained after the development of virus-specific primers, conserved amino acids, followed by the replicase of whereas the 5’ end of the genome was obtained using a Botrytis virus X (BVX, genus Botrexvirus) and members of RLM-RACE Kit (Ambion) [15]. The genome of the virus the genus Potexvirus (potato virus X, plantago asiatica was then re-sequenced from numerous clones generated by mosaic virus, hosta virus X, etc,), with *35-40% amino RT-PCR, employing virus-specific primers in order to acid conservation. However, the BVE polyprotein shares a ensure at least 5x sequence coverage. higher level of aa identity (79%) with the partial sequences Sequence data were assembled with Lasergene soft- of helicase-polymerase domains of cassia mild mosaic ware (DNA Star Inc.) and compared to available virus (accession no. GU481094), an unassigned member of sequences present in the NCBI database using BLASTx the family Alphaflexiviridae. and BLASTp [2]. The open access resources CDD [10] ORF2 codes for a putative 246-aa protein with a predicted and Phobius [8] were used for identification and analysis Mr of 27.3 kDa (p27). The putative protein contains NTP- of conserved motifs in putative genome products. Clu- binding motifs characteristic of helicases (GxxGxGKT/ST) stalW [18] and the neighbour-joining method [16] were [5] that are present in the triple gene block protein 1 (TGBp1) used to align BVE sequences with members of the fam- of members of the families Alphaflexiviridae and Betaflexi- ilies Alphaflexiviridae and Betaflexiviridae and determine viridae [11]. The protein shares 44-46% aa identity and their relationships. Phylogenetic trees were visualized 58-63% similarity with allexivirus orthologs, whereas it is with TreeView [14]. less closely related to foveavirus and potexvirus orthologs Sequence analysis confirmed that a new virus, provi- (30-34% identity and 47-50% similarity with foveaviruses sionally named blackberry virus E (BVE), infects black- and *30% identity/similarity with potexviruses). Orthologs berry. The complete genome of BVE, excluding the 3’ of the protein are known suppressors of RNA silencing poly-A tract, is 7,718 nt long, with an overall A?U content [3, 23]. of 47%. The 97-nt-long 5’untranslated region (UTR) starts ORF3 (nt 5342-5656) codes for a 104-aa-long protein with the pentanucleotide motif ‘‘GAAAA’’, which is con- with an estimated Mr of 11.5 kDa (p12) that has two served in genomes of numerous flexiviruses, followed by transmembrane domains at residues 12-31 and 70-89 [8], five open reading frames (ORFs) (Fig. S1). features that are shared with TGBp2 orthologs of allexi-, 123 Blackberry virus E characterization 1667 fovea-, potex-, and carlaviruses (plant viral movement observed when analyses were performed on other putative proteins pfam_01307). genome products, including the CP (data not shown). Instead of the ‘‘classic’’ TGBp3-encoding ORF, BVE BVE-encoded proteins shared identities ranging from has a 110-codon-long putative ORF (designated ORFx) *25% (ORF4) to *60% (ORF1) with the corresponding between nt 5454 and 5786, which lacks an AUG initiation products of members of recognized species in the genus codon. In silico translation of this ORF showed that the Allexivirus (Table S2). Regardless of the gene used for C-terminal half of the putative protein shares 30-35% comparisons, the levels of identity between BVE and any identical amino acids with the TGBp3 proteins encoded by recognized allexivirus were always significantly lower than the potexviruses mint virus X (MVX), lily virus X (LVX) the identities among allexiviruses. Furthermore, unlike all and alstroemeria virus X (AlVX). Furthermore, this puta- extant allexiviruses, which have a uniform genomic orga- tive protein contains a hallmark motif, CX(5)GX(6-9)C, that nization, BVE lacks the 3’-end-proximal ORF (p15), which is present in TGBp3s of viruses belonging to different encodes a nucleic-acid-binding protein (NABP). BVE also genera in the families Alphaflexiviridae and Betaflexiviri- differs from extant allexiviruses in its natural host range. dae (Fig. 2A and B). Allexiviruses have been reported exclusively from mono- ORF 4 encodes a protein of 356 aa with a Mr of 40 kDa, cots and have a very restricted natural host range consisting which is the least conserved protein in the BVE genome. of members in the genus Allium, whereas BVE was found The serine-rich 40K protein contains