Molecular characterization of a novel rhabdovirus infecting blackcurrant identified by high-throughput sequencing
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 . 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  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.
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.
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Conflict of interest
All authors declare that they have no conflict of interest.
This article does not contain any research involving human or animal participants.
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