Arch Virol DOI 10.1007/s00705-014-2254-5

ANNOTATED SEQUENCE RECORD

Complete genome sequence of motherwort yellow mottle virus, a novel putative member of the genus Torradovirus Jang-Kyun Seo • Minji Kang • Hae-Ryun Kwak Mi-Kyeong Kim • Chang-Seok Kim • Su-Heon Lee • Jeong-Soo Kim • Hong-Soo Choi

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Received: 7 August 2014 / Accepted: 30 September 2014 Ó Springer-Verlag Wien 2014

Abstract The complete genome sequence of a new virus isolated from a motherwort plant exhibiting yellow mottle, mild mosaic, and stunting symptoms in Andong, Korea, was determined. The genome of this virus is composed of two single-stranded RNAs (7068 and 4963 nucleotides in length, respectively) carrying poly(A) tails. RNA1 contains one large open reading frame (RNA1-ORF1), while two potential ORFs (RNA2-ORF1 and RNA2-ORF2) were found in RNA2. BLAST searches of protein databases showed that RNA1-ORF1 and RNA2-ORF2 have maximum amino acid sequence identities of 53 % and 57 % to the RNA1-ORF1 and RNA2-ORF2, respectively, of lettuce necrotic leaf curl virus (LNLCV, a recently identified torradovirus). Phylogenetic analysis provided further evidence that the virus identified in this study is probably a member of a new species in the genus Torradovirus. The name ‘‘motherwort yellow mottle virus’’ (MYMoV) is proposed for this new virus.

J.-K. Seo (&)  M. Kang  H.-R. Kwak  M.-K. Kim  C.-S. Kim  H.-S. Choi (&) Crop Protection Division, National Academy of Agricultural Science, Rural Development Administration, Jeonju 565-851, Republic of Korea e-mail: [email protected] H.-S. Choi e-mail: [email protected] S.-H. Lee School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Republic of Korea J.-S. Kim Department of Plant Medicine, Andong National University, Andong 760-749, Republic of Korea

According to the Virus Taxonomy released by the International Committee on Taxonomy of Viruses (ICTV) in 2013, the genus Torradovirus, family Secoviridae, order Picornavirales, is the taxonomic home of two viruses of two separate viral species: tomato torrado virus (ToTV) and tomato marchitez virus (ToMarV) [1, 7–9]. In addition to the two viruses mentioned above, tomato chocolate virus (ToChV), tomato chocolate spot virus (ToCSV), and lettuce necrotic leaf curl virus (LNLCV) have been identified recently and proposed as members of new species in the genus Torradovirus [3, 10, 11]. The genome of ToTV, a member of the type species of the genus Torradovirus, includes two single-stranded RNAs of *7.8 and *5.4 kilonucleotides. RNA1 contains one large open reading frame (ORF) predicted to encode a polyprotein carrying replication-related motifs. RNA2 contains two potential ORFs encoding proteins of 20 and 134 kDa [8]. Leonurus sibiricus L., commonly called motherwort, is an herbaceous biennial plant in the mint family, Lamiaceae. Motherwort has been commercially cultivated on a small scale in Korea because it has been widely used as a traditional medicinal herb to treat various gynecologic diseases [4]. Since motherwort is also a native weed plant in Asia, it is widely distributed and commonly found near crop fields in Korea. Only a few viruses, including tomato yellow spot virus have been identified in motherwort [2]. In August 2012, field-grown motherwort plants showing yellow mottle, mild mosaic, and stunting symptoms were collected near a pepper field in Andong, Korea. To identify causal agent(s), total RNA was isolated from symptomatic leaves using a PureLinkTM RNA Mini Kit (Invitrogen, Carlsbad, CA) and used to generate a transcriptome library using a TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina, San Diego, CA) following the manufacturer’s protocol. Next-generation sequencing (NGS) was

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J.-K. Seo et al. Table 1 Comparison of amino acid sequences of the genes encoded by motherwort yellow mottle virus with those of other members of the genus Torradovirus

Virus

Acronym

Amino acid sequence identity (%) RNA1OFR1

Protpol

RNA2ORF1

RNA2ORF2

MP

CPs

Lettuce necrotic leaf curl virus

LNLCV

KC855266/ KC855267

53.1

70.4

42.9

55.6

37.4

68.6

Tomato chocolate spot virus

ToChSV

GQ305131/ GQ305132

33.6

57.6

33.2

31.8

18.5

38.8

Tomato chocolate virus

ToChV

FJ560489/ FJ560490

33.6

56.3

30.7

30.6

19.0

37.7

Tomato marchitez virus

ToMarV

EF681764/ EF681765

33.6

58.5

34.7

30.4

18.7

38.2

Tomato torrado virus

ToTV

DQ388879/ DQ388880

32.7

55.2

30.5

30.0

13.9

38.9

performed using an Illumina HiSeq2000 sequencer. De novo assembly of the quality-filtered NGS reads was performed using the Trinity pipeline, and assembled contigs were analyzed by BLASTn and BLASTx searches against the viral reference genome database in GenBank [5]. The entire NGS procedure was performed by Macrogen Inc. (Seoul, South Korea). Among the assembled contigs, two large contigs (7023 bp and 4906 bp in length, respectively) were of virus origin. BLASTx analysis revealed that the first contig contains one large open reading frame (ORF) that has a maximum amino acid sequence identity of 53 % (with 93 % coverage) to the polyprotein (GenBank accession no. AGR55590) encoded by LNLCV RNA1 (KC855266), and the second contig harbors an ORF that has a maximum amino acid sequence identity of 57 % (with 70 % coverage) to the ORF2 (AGR55592) of LNLCV RNA2 (KC855267). To confirm the NGS results, the full-length contigs were amplified by reverse transcription polymerase chain reaction (RT-PCR) using specific primers (50 TTGAAGAACCTACGTCGTTCT-30 and 50 -TCCGA TCTTGCACCTTATTTATTTTA-30 for amplification of the first contig and 50 -GTCGCGCGATTCAACGGTAG-30 and 50 -TCCGATCTTGCACCTTATTTATTTTA-30 for amplification of the second contig) and sequenced by the Sanger method using primers designed based on the NGS results. To obtain complete genome sequences, terminal sequences of each contig were determined by the 50 and 30 rapid amplification of cDNA ends (RACE) method using a SMARTer RACE cDNA Amplification Kit (Clontech, USA) according to the manufacturer’s instruction. Analysis of the 30 end sequences revealed that both contigs contained poly(A) tails at the 30 ends. The assembled fulllength sequences of the contigs were 7068 and 4963 nucleotides (nt) in length, respectively, excluding the poly(A) tail. Low-stringency nucleotide–nucleotide BLAST analyses indicated that the first and second contigs

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GenBank no. (RNA1/RNA2)

have maximum nucleotide sequence identities of 68 % (with 44 % coverage) and 67 % (with 31 % coverage) to LNLCV RNA1 (KC855266) and RNA2 (KC855267). Therefore, we concluded that the full-length sequences of the two contigs represent the full genome (RNA1 and RNA2) of a novel putative member of the genus Torradovirus, and the name ‘‘motherwort yellow mottle virus’’ (MYMoV) is proposed for this new torradovirus. The fulllength sequences of MYMoV RNA1 and RNA2 were deposited in GenBank under the accession numbers KM229700 and KM229701, respectively. The genome organization of MYMoV is analogous to those of other torradoviruses [8, 9, 11]. MYMoV RNA1 contains one large ORF (RNA1-ORF1) of 6573 nt, encoding a predicted polyprotein (245 kDa) carrying replication-related motifs. The 50 and 30 untranslated regions (UTRs) of RNA1 are 135 and 360 nt in length, respectively. MYMoV RNA2 contains two ORFs (RNA2ORF1 and RNA2-ORF2). RNA2-ORF1 encodes a functionally unknown protein (23 kDa), and RNA2-ORF2 encodes a predicted polyprotein (134 kDa) that is predicted to be cleaved to yield the putative movement protein (MP) and three distinct coat proteins (CPs). The 50 and 30 UTRs of RNA2 are 406 and 464 nt in length, respectively. To examine the taxonomic position of MYMoV, phylogenetic analysis and sequence comparisons were performed as described in previous studies reporting new torradoviruses [3, 10, 11]. First, the amino acid region between the CG protease motif and the GDD RNA replicase motif (Prot-pol) in the RNA1-ORF1 of MYMoV was phylogenetically compared with those of other torradoviruses and several members of the family Secoviridae. A phylogenetic tree was reconstructed by the maximumlikelihood (ML) method implemented in the MEGA 5.1 program using an amino acid sequence alignment generated by the Clustal X program [6]. The resulting

Genome sequence of motherwort yellow mottle virus

Fig. 1 Phylogenetic relationship of motherwort yellow mottle virus to other members of the family Secoviridae. Phylogenetic trees were constructed using the maximum-likelihood (ML) method with the MEGA 5.1 program based on an alignment of the region between the protease CG motif and the GDD RNA replicase motif (A) or fulllength nucleotide sequences of RNA1 (B) or RNA2 (C) of the indicated viruses. The numbers on the branches indicate bootstrap percentages based on 1000 replications (only values [60 % are shown). GenBank accession numbers of the virus sequences included in the analysis are as follows: apple latent spherical virus (ALSV; NC003787(RNA1)/NC003788(RNA2)), carrot necrotic dieback virus

(CNDV; EU980442), cherry rasp leaf virus (CRLV; NC006271), lettuce necrotic leaf curl virus (LNLCV; KC855266(RNA1)/ KC855267(RNA2)), maize chlorotic dwarf virus (MCDV; NC003626), parsnip yellow fleck virus (PYFV; NC003628), potato virus Y (PVY; EF026076), rice tungro spherical virus (RTSV; NC001632), tomato chocolate spot virus (ToChSV; GQ305131(RNA1)/GQ305132(RNA2)), tomato chocolate virus (ToChV; FJ560489(RNA1)/FJ560490(RNA2)), tomato marchitez virus (ToMarV; EF681764(RNA1)/EF681765(RNA2)), and tomato torrado virus (ToTV; DQ388879(RNA1)/DQ388880(RNA2))

phylogenetic analysis showed that MYMoV is most closely related to LNLCV and clusters together with the other members of the genus Torradovirus (Fig. 1A). Other phylogenetic analyses based on full-length nucleotide sequences of RNA1 (Fig. 1B) and RNA2 (Fig. 1C) consequently supported assignment of MYMoV to this genus. However, the overall levels of identity between MYMoV

and other known torradoviruses are significantly low (Table 1). Especially, the amino acid sequence identities of the Prot-pol and CPs between MYMoV and other torradoviruses were in the range of 55.2–70.4 % and 37.7–68.6 %, respectively (Table 1). Therefore, our results suggest that MYMoV is a member of a new species in the genus Torradovirus. Further studies are in progress to

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examine the biological features of MYMoV and to evaluate its potential impact on agriculture.

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Acknowledgments This research was supported by a grant from the Agenda Program (PJ008841) funded by the Rural Development Administration of Korea.

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