Archives of Virology

, Volume 163, Issue 5, pp 1363–1366 | Cite as

Molecular characterization of a novel rhabdovirus infecting blackcurrant identified by high-throughput sequencing

Annotated Sequence Record

Abstract

A large contig with sequence similarities to several nucleorhabdoviruses was identified by high-throughput sequencing analysis from a black currant (Ribes nigrum L.) cultivar. The complete genome sequence of this new nucleorhabdovirus is 14,432 nucleotides long. Its genomic organization is very similar to those of unsegmented plant rhabdoviruses, containing six open reading frames in the order 3′-N-P-P3-M-G-L-5. The virus, which is provisionally named “black currant-associated rhabdovirus”, is 41-52% identical in its genome nucleotide sequence to other nucleorhabdoviruses and may represent a new species in the genus Nucleorhabdovirus.

The family Rhabdoviridae includes 131 recognized viruses in 18 genera that infect a wide range of organisms, including vertebrates, invertebrates and plants [1]. These viruses have enveloped bullet-shaped (vertebrates and invertebrates) or bacilliform particles (plants) with a non-segmented, negative-sense, single-stranded RNA genome of 11–15 kb. Plant rhabdoviruses are classified into four genera, Cytorhabdovirus, Dichorhavirus, Nucleorhabdovirus and Varicosavirus, based on their intracellular site of replication, genome structure and vector species [2, 3]. Rhabdoviruses infect a wide range of monocots and dicots and are mainly transmitted by planthoppers, leafhoppers, aphids, mites and fungi. To date, ten nucleorhabdoviruses have been described, all from poaceous crops and herbaceous plants [2, 3, 4, 5]. Rhabdovirus-like particles of 65-80 × 215-485 nm have been found in both the nucleus and cytoplasm of phloem cells of blackcurrant plants with symptoms of blackcurrant reversion and gooseberry vein-banding diseases [6, 7, 8]. However, nucleotide sequences of any of these rhabdoviruses are not currently available.

An asymptomatic blackcurrant (Ribes nigrum L.) cv. Veloy accession was used as a negative control when a symptomatic accession cv. Malochka was subjected to high-throughput sequencing (HTS) for pathogen identification. The accession cv. Malochka plants senesced early, and all leaves were gone by early July, when all other blackcurrant accessions were full of green leaves. Both accessions were negative by virus-specific RT-PCR/PCR assays for blackcurrant reversion virus (BRV), gooseberry vein banding virus and tomato ringspot virus. Total RNA was isolated from leaf tissue using an RNeasy Plant Mini Kit (QIAGEN, MD) and subjected to Illumina NextSeq 500 sequencing (SeqMatic, CA). Plant ribosomal RNA (rRNA) was removed using an Illumina Ribo-Zero rRNA Removal Kit before cDNA library construction. Analysis of total RNA reads was performed using CLC Genomics Workbench 9.5.2. Contigs were annotated by Blastx comparisons. The virus genome was re-sequenced using plasmid DNA cloned from RT-PCR products amplified using virus-specific primers (Supplementary Table 1). The exact ends of the genome were determined using a 5′ and 3′ RACE system for Rapid Amplification of cDNA Ends (Life Technologies). The sequence of each amplicon was determined from at least five clones from both directions. Phylogenetic analysis was conducted using MEGA7.1. To screen for the virus in other plants, total nucleic acid was extracted using a CTAB method [9] and tested by RT-PCR.

After removal of failed and low-quality reads, 37,200,461 and 38,139,849 75-bp paired reads were obtained from the asymptomatic and symptomatic accessions, respectively. De novo assembly of RNA reads generated 44,104 and 41,369 contigs (> 200 nt) from the asymptomatic and symptomatic samples, respectively. A Blastx search of the contigs against the Viruses_NR database revealed a large contig (14,420 nucleotides [nt]) with the highest amino acid (aa) sequence identity of 50% with datura yellow vein virus (DYVV) (GenBank no. NC_028231) in the asymptomatic accession. A total of 2,676,391 reads (185x coverage) mapped to this contig, supporting its presence in the accession. No other virus, viroid or phytoplasma was identified. The presence of this new virus was confirmed by RT-PCR using virus-specific primers hC812f1 and hC812r1 (Supplementary Table 1). The virus did not induce any obvious symptoms in the host in which it was detected, and it is provisionally named “black currant-associated rhabdovirus” (BCaRV). Eight contigs (405-5,056 nt) with aa sequence identities of 29-49% to a number of closteroviruses were identified from the symptomatic accession.

The complete sequence of the BCaRV genome is 14,432 nt (GenBank accession no. MF543022). The 3’ leader and 5’ trailer sequences of BCRV-1 share 17/21 complementary nucleotides, forming a putative handle structure (Supplementary Fig. 1A). The genome organization of the virus is very similar to those of unsegmented plant rhabdoviruses, containing six open reading frames (ORFs) in order of 3’-N (nucleocapsid protein, 468 aa) – P (phosphoprotein, 326 aa) – P3 (putative movement protein, 322 aa) – M (matrix protein, 276 aa) – G (glycoprotein 641 aa) – L (polymerase, 2106 aa) -5’ in negative polarity (Fig. 1A) [1, 2, 3, 10]. These ORFs are separated by conserved intergenic regions, which are composed of three elements (Supplementary Fig. 1B) [4, 11]. The intergenic sequences of BCaRV are identical to those of DYVV and sonchus yellow net virus (SYNV) (Supplementary Fig. 1C) [2, 4, 11, 12]. Sequence analysis using cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) showed that all BCaRV-encoded proteins contain a classical mono- or bipartite nuclear localization signal (NLS) [2, 4, 11, 12]. The scores obtained using cNLS Mapper predicted exclusive nuclear localization for the N protein, partial nuclear localization for the M, G and L proteins, and both cytoplasmic and nuclear localization for the P and P3 proteins (Fig. 1B) [5, 10]. A leucine-rich nuclear export signal was predicted using NetNes [5, 9, 11] in all encoded proteins with the exception of the N protein (Fig. 1B).
Fig. 1

A) Schematic representation of the genome organization of black currant-associated rhabdovirus (BCaRV) and B) percentage sequence identities of the genome and putative gene products between BCaRV and selected members of the family Rhabdoviridae. cNLS Mapper score, a cell nuclear localization signal prediction program; nd, not detected; +, detected; A cNLS score of 8, 9, or 10 predicts that the protein is exclusively localized to the nucleus, a score of 7 or 8 indicates partial localization to the nucleus, a score of 3, 4, or 5 indicates localization to both the nucleus and the cytoplasm, and a score of 1 or 2 indicates localization to the cytoplasm. DYVV, datura yellow vein virus NC_028231); SYNV, sonchus yellow net virus (NC_001615); PYDV, potato yellow dwarf virus (NC_016136); OFV, orchid fleck virus (NC_009608) (a dichorhavirus); LNYV, lettuce necrotic yellows virus (NC_007642) (a cytorhabdovirus)

Comparisons of the genomic sequences of BCaRV and other rhabdoviruses showed that BCaRV is most similar to nucleorhabdoviruses (Fig. 1B). Phylogenetic analysis based on the aa sequences of the L protein showed that BCaRV is most closely related to SYNV, DYVV, maize fine streak virus (MFSV), and the dichorhaviruses, and together, they form a cluster distinct from that of other nucleorhabdoviruses (Fig. 2). The topologies of the phylogenetic trees constructed using aa sequences of the putative proteins N, P, P3 and G differed slightly, but the close relationship of the virus to DYVV and SYNV remained (data not shown). The three viruses grouped with MFSV together in a distinct cluster within the genus Nucleorhabdovirus when the N, P and G proteins were analyzed, whereas the P3 proteins of the three viruses were more closely related to those of the cytorhabdoviruses. Taken together, these results strongly support the conclusion that BCaRV is a new nucleorhabdovirus.
Fig. 2

Neighbor-joining tree constructed from deduced amino acid sequences of the polymerase (L protein) of black currant-associated rhabdovirus (BCaRV and selected plant rhabdoviruses. Bootstrap analysis was applied using 1000 replicates. A solid diamond indicates BCaRV

A total of 38 germplasm accessions representing different Ribes species (black currant, red currant and gooseberry) from the National Clonal Germplasm Repository in Oregon were tested by RT-PCR using the primers hc812f2 and hc812r2 (Supplementary Table 1). BCaRV was detected in two black currant accessions. One was the second copy of cv. Veloy, and the other was cv. Burga from France (Supplementary Fig. 2). The cv. Burga plant showed foliar chlorosis and was also infected with BRV. A 1,348-bp amplicon of the Burga isolate was cloned and sequenced. The results showed that the two isolates were 80% identical to each other at the nucleotide sequence level in the N-G region, suggesting high genetic divergence among isolates.

The nucleotide sequence identities of 41-52% between BCaRV and ten other nucleorhabdoviruses fall into the range of 41-54% among known nucleorhabdoviruses. Therefore, BCaRV should be considered a member of a new virus species in the genus Nucleorhabdovirus. The 14,432-nt genome of BCaRV is the largest among the nucleorhabdoviruses due to a longer 5’-nontranslated region and the fact that most of the intergenic regions are longer. An asymptomatic host may serve as a reservoir for the virus, and mixed infection with other viruses may cause disease in the host plants. The genomic sequence obtained in this study is valuable for detection and further characterization of BCaRV.

Notes

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any research involving human or animal participants.

Supplementary material

705_2018_3709_MOESM1_ESM.docx (27 kb)
Supplementary material 1 (DOCX 29 kb)
705_2018_3709_MOESM2_ESM.pptx (174 kb)
Supplementary material 2 (PPTX 173 kb)
705_2018_3709_MOESM3_ESM.docx (14 kb)
Supplementary material 3 (DOCX 13 kb)
705_2018_3709_MOESM4_ESM.txt (15 kb)
Supplementary material 3 (TXT 13 kb)

References

  1. 1.
    Amarasinghe GK, Bào Y, Basler CF, Bavari S, Beer M, Bejerman N, Blasdell KR, Bochnowski A, Briese T, Bukreyev A, Calisher CH, Chandran K, Collins PL, Dietzgen RG, Dolnik O, Dürrwald R, Dye JM, Easton AJ, Ebihara H, Fang Q, Formenty P, Fouchier RAM, Ghedin E, Harding RM, Hewson R, Higgins M, Hong J, Horie M, James AP, Jiāng D, Kobinger GP, Kondo H, Kurath G, Lamb RA, Lee B, Leroy EM, Li M, Maisner A, Mühlberger E, Netesov SV, Nowotny N, Patterson JL, Payne SL, Paweska JT, Pearson N, Randall RE, Revill PA, Rima BK, Rota P, Rubbenstroth D, Schwemmle M, Smither SJ, Song Q, Stone DM, Takada A, Terregino C, Tesh RB, Tomonaga K, Tordo N, Towner JS, Vasilakis N, Volchkov VE, Wahl-Jensen V, Walker PJ, Wang B, Wang D, Wang F, Wang L-F, Werren JH, Whitfield AE, Yan Z, Ye G, Kuhn JH (2017) Taxonomy of the order Mononegavirales: update 2017. Arch Virol 162:2493–2504CrossRefPubMedGoogle Scholar
  2. 2.
    Dietzgen RG, Kondo H, Goodin MM, Kurath G, Vasilakis N (2017) The family Rhabdoviridae: mono- and bipartite negative-sense RNA viruses with diverse genome organization and common evolutionary origins. Virus Res 227:158–170CrossRefPubMedGoogle Scholar
  3. 3.
    Jackson AO, Dietzgen RG, Goodin MM, Bragg JN, Deng M (2005) Biology of plant rhabdoviruses. Annu Rev Phytopathol 43:623–660CrossRefPubMedGoogle Scholar
  4. 4.
    Pappi PG, Dovas CI, Efthimiou KE, Maliogka VI, Katis NI (2013) A novel strategy for the determination of a rhabdovirus genome and its application to sequencing of Eggplant mottled dwarf virus. Virus Genes 47:105–113CrossRefPubMedGoogle Scholar
  5. 5.
    Dietzgen RG, Innes DJ, Bejerman N (2015) Complete genome sequence and intracellular protein localization of Datura yellow vein nucleorhabdovirus. Virus Res 205:7–11CrossRefPubMedGoogle Scholar
  6. 6.
    Boyko AL, Spaar D, Polischuk VP, Senchugova NA, Mishenko LT, Silajeva AM, Glushak LE, Taranuho NP (1995) The exploration of rhabdoviruses infecting agricultural plants in conditions of the Ukraine. Arch Phytopathol Pflanz 30:85–90CrossRefGoogle Scholar
  7. 7.
    Roberts IM, Jones AT (1997) Rhabdovirus-like and closterovirus-like particles in ultrathin sections of Ribes species with symptoms of blackcurrant reversion and gooseberry vein banding diseases. Ann Appl Biol 130:77–89CrossRefGoogle Scholar
  8. 8.
    Pribylova J, Spak J, Kubelkova D (2002) Mixed infection of black currant (Ribes nigrum L.) plants with blackcurrant reversion associated virus and rhabdovirus-like particles with symptoms of black currant reversion disease. Acta Virol 46:253–256PubMedGoogle Scholar
  9. 9.
    Li R, Mock R, Huang Q, Abad J, Hartung J, Kinard G (2008) A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. J Virol Methods 154:48–55CrossRefPubMedGoogle Scholar
  10. 10.
    Kosugi S, Hasebe M, Tomita M, Yanagawa H (2009) Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc Natl Acad Sci USA 106:10171–10176CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Cour TL, Kiemer L, Mølgaard A, Gupta R, Skriver K, Brunak S (2004) Analysis and prediction of leucine-rich nuclear export signals. Protein Eng Des Sel 17:527–536CrossRefPubMedGoogle Scholar
  12. 12.
    Heaton LA, Hillman BI, Hunter BG, Zuidema D, Jackson AO (1989) Biochemistry Physical map of the genome of Sonchus yellow net virus, a plant rhabdovirus with six genes and conserved gene junction sequences. Proc Natl Acad Sci USA 86:8665–8668CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© This is a U.S. government work and its text is not subject to copyright protection in the United States; however, its text may be subject to foreign copyright protection 2018

Authors and Affiliations

  • L.-P. Wu
    • 1
    • 2
  • T. Yang
    • 2
  • H.-W. Liu
    • 1
  • J. Postman
    • 3
  • R. Li
    • 1
  1. 1.USDA-ARS, National Germplasm Resources LaboratoryBeltsvilleUSA
  2. 2.Key Laboratory of Poyang Lake Environment and Resource, School of Life ScienceNanchang UniversityNanchangChina
  3. 3.USDA-ARS, National Clonal Germplasm RepositoryCorvallisUSA

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