Nepoviruses (genus Nepovirus, family Secoviridae, suborder Comovirinae, order Picornavirales) are small icosahedral viruses (ca 28 nm in diameter) with a bipartite positive–strand, poly-A tailed RNA genome. Each of the two genomic RNA encodes a single large polyprotein. RNA 1 ranges in size from 7,2 kb to 8,4 kb and encodes the replication-related proteins. RNA 2 ranges in size from 3,7 kb to 7,3 kb and encodes the coat protein (CP) of 55-60 kDa and the movement protein (MP). Based on the phylogenetic relationships of the CPs, specific size of RNA 2, and cleavage site specificity of the viral protease, nepoviruses are divided into three subgroups (A, B and C) [1].

Beet ringspot virus (BRSV) was originally isolated in Scotland as a soil-borne virus, infecting a wide range of plant species including sugar beet, potato, turnip, wheat, oat, strawberry, many weeds and peach [2]. It was later thought to be a serotype of tomato black ring virus (TBRV-S) but based on the RNA sequence data the ICTV reclassified this virus as a distinct species within subgroup B of the nepoviruses [3]. This study deals with the molecular characterization of the first Hungarian isolate of BRSV (BRSV-Br1) discovered in a new natural host Begonia ricinifolia that showed unusual ring spot and line pattern symptoms (Fig.1A).

Fig. 1
figure 1

(A) Ringspot and line pattern symptoms in B. ricinifolia infected by BRSV-Br1. (B) Transmission electron micrograph of negatively stained BRSV-Br1 particles. (C) PAGE analysis of the BRSV-Br1 coat protein

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The Br1 isolate was transferred from symptomatic B. ricinifolia to Nicotiana benthamiana by sap inoculation and virions were purified from systemically infected N. benthamiana leaves [4]. Purified virions negatively stained with 2% uranyl acetate showed spherical particles ca. 30 nm in diameter with hexagonal outlines when examined by transmission electron microscopy (JEOL JEM-1011) (Fig. 1B). Proteins were prepared from virions [5] and separated by electrophoresis in 12% TGX Stain-Free™ FastCast™ Acrylamide Gels (Bio-Rad) in the presence of ProSieve QuadColor™ protein marker (4.6 kDa – 300 kDa) (Lonza). A single protein molecule of ca. 56 kDa characteristic of nepovirus CPs was visualized (Fig. 1C).

Total RNA was extracted from the diseased leaves of N. benthamiana using a phenol-chloroform extraction method [6] and used for first-strand cDNA synthesis with the High-Capacity cDNA reverse transcription kit (Applied Biosystems) with the primer oligo(dT) 18 following the manufacturer’s protocols.

The complete nucleotide sequences of RNA 1 and RNA 2 (accession number MF141079 and MF141080, respectively) were determined using specific primers derived from the BRSV reference genome sequences deposited in the NCBI database (NC_003693 and NC_003694, respectively). The regions used for the design of specific primers were re-amplified and sequenced to rule out the presence of mutations. The 5’- terminal sequences of RNA 1 and RNA 2 were determined using 5’ FirstChoice RLM-RACE kit (Ambion) with virus specific primers. All PCR amplifications were performed with high-fidelity Phusion DNA polymerase (Thermo Fisher Scientific) to ensure that the authentic genome sequence of BRSV-Br1 was obtained. Computer based analysis of the viral genome (Fig. 2A) and phylogenetic analyses (Fig. 2B) were conducted using the EMBOSS software package [7] and MEGA 7 [8], respectively.

Fig. 2
figure 2

(A) Schematic representation of the genome organization of BRSV-Br1. Grey boxes indicate ORFs, with the names of predicted gene products and Mrs below them. Polyprotein putative cleavage sites that release the specific proteins are indicated (dotted lines) with the sequence of the cleaved dipeptide. The positions of the identified conserved motifs are represented by striped boxes. The putative cleavage sites were deduced by the similarity of the dipeptide alongside the upstream sequence using previously identified or inferred sites from other nepoviruses, fitting the taxonomy demarcation of subgroup B of nepoviruses. Question marks in RNA 2 means that we were unable to identify the exact putative cleavage site to separate 2A from MP and its location and Mrs is an estimation. (B) Maximum-likelyhood phylogenetic tree showing the relationship of BRSV-Br1 and other nepoviruses based on alignments of CPs. Branch lengths are proportional to the genetic distances. Cymbidium ringspot virus (genus Tombusvirus) was used as outgroup. Numbers on branches indicate percantage of bootstrap support out of 1000 bootstrap replications. Bootstrap percentages greater than 50 % are shown

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BRSV-Br1 RNA 1 is 7394 nt long and contains a single 6810 nt long ORF, which is translated into a 2269 amino acid long polyprotein with a molecular weight of 254,6 kDa. The RNA 1 encoded polyprotein (P1) is predicted to be cleaved by the viral proteinase into five mature proteins. These were identified based on the conserved sequence motifs [1] as the: proteinase cofactor (Pro-cof), NTP binding protein (NTP-b), viral genome-linked protein (VPg), proteinase (Pro) and RNA-dependent RNA polymerase (RdRp) (Fig. 2A). The complete sequence of RNA 2 is 4684 nt long. It contains a 4053 nt long ORF which encodes a single polyprotein of 1350 amino acids with a molecular weight of 149 kDa. Based on homology to other subgroup B nepoviruses [1], the RNA 2 encoded polyprotein (P2) is predicted to be processed into mature protein 2A, the putative movement protein (MP) and the coat protein (CP) (Fig. 2A).

Sequence comparison of P1 and P2 with other members of the genus Nepovirus, revealed that the isolate Br1 show 94% and 92 % identity with the corresponding polyproteins of BRSV. Phylogenetic analysis of the predicted amino acid sequence of the putative CP also confirmed that it is most closely related to BRSV and belongs to subgroup B of the genus Nepovirus (Fig. 2B). Based on the molecular and phylogenetic data we conclude that isolate Br1 is a strain of BRSV and propose to name it BRSV-Br1.