KRAUSE-SAKATE, R.

MARCHI, B. R. de

NOZAKI, D. N.

PAVAN, M. A.

SANCHES, M. M.

SPADOTTI, D. M.

English

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ANNOTATED SEQUENCE RECORDBiological and molecular characterisation of Bidens mosaic virussupports its assignment as a member of a distinct speciesin the genus PotyvirusMarcio Martinello Sanches • Bruno R. De Marchi •David Marques Spadotti • Denise Nakada Nozaki •Marcelo A. Pavan • Renate Krause-SakateReceived: 18 October 2013 / Accepted: 3 March 2014 / Published online: 15 March 2014 Springer-Verlag Wien 2014Abstract The complete nucleotide (nt) sequence of Bi-dens mosaic virus (BiMV) isolate SP01 was determinedand shown to consist of 9,557 nt. Since it shared highestidentities in the nt sequence of the whole genome(66–73 %) and in the aa sequence of the polyprotein(60–76 %) with viruses of the potato virus Y subgroup, itwas compared with them genetically and biologically.Phylogenetic analysis showed that the closest relative ofBiMV is sunflower chlorotic mottle virus, from which it,however, differed significantly in various respects. Theseresults indicate that BiMV should represent a distinctspecies in the genus Potyvirus.Bidens mosaic virus (BiMV) was described in 1961 [1] as avirus causing mosaic symptoms in Bidens pilosa L. andbelonging to the genus Potyvirus. This virus is apparentlyrestricted to Brazil and was reported to naturally infect B.pilosa, Helianthus annuus [2], Coreopsis lanceolata [3],Pisum sativum [4], Lactuca sativa [5], and Galingsogaparviflora [6]. Although BiMV was initially considered aseparate potyvirus [1–5], it is currently classified as a strainof potato virus Y (PVY) [7] based on its coat protein (CP)gene sequence [8]. In order to clarify the taxonomic statusof BiMV, we sequenced the complete genome of an isolateof the virus.The isolate SP01 of BiMV was collected from infectedB. pilosa plants found in Botucatu, São Paulo State, Brazil,in 2008. It was then sap transmitted in a host rangeexperiment. Total RNA was extracted from infected Che-nopodium quinoa leaves using a Total RNA PurificationKit (Norgen Biotek). The cDNA was prepared with apoly(T) primer and with the primers CIrev and HPrev [9],using AMV reverse transcriptase (Promega) according tothe manufacturer’s instructions. The CI and HC-Pro geneswere amplified using previously described universalprimers for members of the family Potyviridae [9]. The CPand nuclear B inclusion (NIb) genes were amplified usingthe universal primer WCIEN [10] with the primer 7587[11]. The gaps between these regions were amplified usingspecific primers designed from the previously obtainedsequences. Polymerase chain reactions (PCRs) were per-formed using Platinum high-fidelity Taq DNA polymerase(Invitrogen). The 5’-proximal region of the genomicsequence was determined using the 5’ Rapid Amplificationof cDNA Ends (RACE) (Clontech) following the manu-facturer’s instructions. The amplified fragments werecloned using the pGEM-T Easy Vector system (Promega).For each fragment, three independent clones weresequenced in both directions at Macrogen Inc. (Republic ofKorea). A consensus sequence was assembled usingDNAMAN 4.0 (Lynnon Corporation), and the final gen-ome sequence was deposited in the DDBJ/EMBL/GenBankdatabase under the accession number KF649336. Com-parisons were made between the BiMV-SP01 sequence andthe available complete sequence of the most closely relatedpotyviruses. Also, the CP gene sequences of two BiMVisolates available from GenBank were compared to BiMV-SP01. The sequences were aligned using ClustalW [12]Electronic supplementary material The online version of thisarticle (doi:10.1007/s00705-014-2047-x) contains supplementarymaterial, which is available to authorized users.M. M. Sanches (&)  B. R. De Marchi  D. M. Spadotti D. N. Nozaki  M. A. Pavan  R. Krause-SakateUNESP-Faculdade de Ciências Agronômicas, Botucatu, Brazile-mail: marcio.sanches@embrapa.brM. M. SanchesEMBRAPA-Recursos Genéticos e Biotecnologia, Brası́lia,Brazil123Arch Virol (2014) 159:2181–2183DOI 10.1007/s00705-014-2047-xwith default parameters. Maximum-likelihood phyloge-netic trees were prepared using MEGA 5.0 [13] with 1,000bootstrap replicates. Recombination analysis was per-formed with the programs GENECONV, RDP, Bootscan,MaxChi, Chimaera, 3Seq, and SiScan packaged in thesoftware RDP3 [14] using default settings.The host range data for BiMV-SP01 were consistentwith those reported for other BiMV isolates that cause localsymptoms in Chenopodium amaranticolor and C. quinoaand systemic infection in Helianthus annus ‘‘Catissol 1’’,Lactuca sativa ‘‘Trocadero’’, Nicotiana benthamiana, N.clevelandii, N. occidentalis, N. rustica, N. tabacum, Pisumsativum and Zinnia elegans [2–4, 15]. BiMV-SP01 alsosystemically infected Gomphrena globosa (symptomless)and Cucurbita pepo, which had not previously beenreported as BiMV hosts. The virus did not infect Ama-ranthus viridis, Capsicum annum ‘‘Magda’’, Chenopodiummurale, Datura metel, D. stramonium, Solanum lycoper-sicum ‘‘Santa Clara’’, Nicandra physaloides, Nicotianaglutinosa, Petunia hybrida, Physalis floridana and Sonchusoleraceus. From symptomatic and symptomless plants,total RNA was extracted as described previously, and thepresence of BiMV was verified by RT-PCR [6].The complete genome of BiMV-SP01 consisted of 9,557nucleotides, excluding the 3’ terminal poly(A) tail. TheAUG codon located at nt position 118–121 is likely to bethe translation initiation codon, since it was in a context(CAAAUGGCA) that is similar to that observed in otherpotyviruses [9]. The 5’ non-coding region (NCR) was 117nt in length, AU-rich, and terminated in several A residues.The 3’ NCR was 256 nt in length and AU-rich. The stopcodon (UGA) was at position 9,301–9,303. The putativetranslation product contained 3,066 amino acids (aa) long,with a calculated molecular mass of 348.2 kDa (http://web.expasy.org/cgi-bin/protparam/protparam). According to theconserved cleavage sites predicted by Adams et al. [16], atotal of 10 proteins are expected: P1, HC-Pro, P3, 6K1, CI,6K2, VPg, NIa, NIb, and CP. The observed cleavage sitesof BiMV-SP01 were perfectly consistent with the knownsites of potyviruses (Supplementary Fig. 1). The putativeopen reading frame (ORF—PIPO) with the G1-2A6-7 motif[17] was found at nucleotides 2847 to 3074 and contained76 aa. The calculated mass of PIPO was 9.1 kDa.When the complete BiMV-SP01 genome and predictedpolyprotein sequences were compared with other sequen-ces of the most closely related potyviruses, it shared thehighest nt (73 %) and aa sequence identities (76 %) withthe C and CRS isolates of sunflower chlorotic mottle virus(SCMoV) (Supplementary Table 1). Nucleotide sequenceidentities of 69–71 % were obtained for the whole genomewhen compared with the sequences of PVY isolates. Theseidentities were below the threshold value that discriminatesbetween potyvirus species and strains according to theninth ICTV report [7]. BiMV-SP01 shared identities of 96and 98 % in the CP nt and aa sequences, respectively, withthe BiMV-p and BiMV-b isolates (accession nos.AY960150 and AY960151).The phylogenetic tree based on multiple alignments ofthe deduced aa sequence of the entire polyprotein (Fig. 1)shows BiMV-SP01 in a separate branch together with thetwo SCMoV isolates. They formed a separate cluster withisolates of potato virus Y (PVY) and pepper severe mosaicvirus (PepSMV), with a bootstrap value of 100 supportingthe existence of an ancestral common origin to the PVY,PepSMV, BiMV, and SCMoV isolates. The Ninth Reportof the International Committee on Taxonomy of Viruses(ICTV) lists BiMV as a strain of PVY and SCMoV as amember of a tentative species in the genus Potyvirus [7];the latter was proposed after the whole genomes of twoFig. 1 Maximum-likelihood tree constructed from the entire poly-protein amino acid sequences of Bidens mosaic virus (BiMV) and themost closely related potyviruses: potato virus Y (PVY), sunflowerchlorotic mottle virus (SCMoV), pepper severe mosaic virus (Pep-SMV), pepper yellow mosaic virus (PepYMV), pepper mottle virus(PepMoV), Peru tomato mosaic virus (PTV), potato virus V (PVV),verbena virus Y (VVY), wild potato mosaic virus (WPMV) andBidens mottle virus (BiMoV). Lettuce mosaic virus (LMV) was usedas an outgroup sequence. The values at the forks indicate thepercentages of 1,000 bootstrap replicates. The corresponding Gen-Bank accession numbers of each virus sequence are given inparentheses. The BiMV isolate is indicated in bold type2182 M. M. Sanches et al.123isolates of SCMoV were sequenced [18]. The recombina-tion analysis of the BiMV-SP01 sequence did not find anyclear signal of a recombination event when aligned to thesequences of viruses belonging to the PVY subgroup.Historically, the taxonomic position of BiMV has beenquestioned. Symptom similarities with another potyvirus,such as Bidens mottle virus (BiMoV), which was foundinfecting lettuce and B. pilosa in the USA [19], and withSCMoV, which was found infecting sunflower and Z. ele-gans in Argentina and Brazil [20, 21], led to proposals thatthey belong to the same species. BiMoV and BiMV infectat least 10 common hosts, and a serological relationshipexists between BiMV and SCMoV [21]. However, whentheir entire genomes were compared, the nt sequenceidentity between BiMV and BiMoV was 67%. Despite thefact that cleavage sites for HC-Pro/P3, 6K1/CI, 6K2/VPg,Vpg/NIa, and NIa/NIb are the same in SCMoV and BiMV,the biological differences in experimental host range [20,21] and the ability of BiMV to naturally infect pea andlettuce [4, 5], including the emergence of a strain withdistinct biological characteristics that causes severesymptoms in the latter host, support the inclusion of BiMVin a distinct species in the genus.Acknowledgments This work was supported by grants receivedfrom the Fundação de Amparo à Pesquisa do Estado de São Paulo(Proc 2007/04162-4).Conflict of interest The authors declare that they have no conflictof interest.References1. Kitajima EW, Carvalho AMB, Costa AS (1961) Morfologia dovı́rus do mosaico do picão. Bragantia 20:503–5122. Costa AS, Kitajima EW (1966) Virus do mosaico do picão atacagirassol. Supl Agric ‘‘O Estado de São Paulo’’, São Paulopp 12–133. 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Biological and molecular characterisation of Bidens mosaic virus supports its assignment as a member of a distinct species in the genus Potyvirus.

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Biological and molecular characterisation of Bidens mosaic virus supports its assignment as a member of a distinct species in the genus Potyvirus.

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