Archives of Virology (2022) 167:973–977

https://doi.org/10.1007/s00705-021-05348-9

ANNOTATED SEQUENCE RECORD

Complete genome sequence of cnidium virus 1, a novel

betanucleorhabdovirus infecting Cnidium officinale
Mesele Tilahun Belete1,2,3 · Davaajargal Igori2,4 · Se eun Kim2 · Su‑Heon Lee5 · Jae Sun Moon1,2 

Received: 10 September 2021 / Accepted: 18 November 2021 / Published online: 3 February 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2022

Abstract
The complete genomic sequence of a plant rhabdovirus that was identified in Cnidium officinale in Yeongyang-dun, South
Korea, is reported here. The virus, tentatively named “cnidium virus 1” (CnV1), has a negative-sense RNA genome
of ~ 14 kb, and its organization most closely resembles that of unsegmented plant rhabdoviruses, containing six antisense
open reading frames (ORFs) in the order 3′-N-P-P3-M-G-L-5′. Intergenic regions containing conserved sequences separate
the genes. The genome of CnV1 is 37.8–56% identical in its complete nucleotide sequence to betanucleorhabdoviruses and
other related rhabdoviruses. Therefore, based on the sequence similarity criteria for species demarcation, its genome organi-
zation, and its phylogenetic position, CnV1 should be classified as a new member of the genus Betanucleorhabdovirus in
the family Rhabdoviridae. CnV1 is the first rhabdovirus found in C. officinale.

In March 2021, the updated ICTV Online (10th) Report [1] Gammanucleorhabdovirus, and Cytorhabdovirus for viruses
showed that the family Rhabdoviridae contains 191 spe- that have an unsegmented genome, and Varicosavirus and
cies in 30 genera, whose members affect a wide range of Dichorhavirus for viruses that have a bisegmented genome,
organisms, including vertebrates, invertebrates, and plants. and based on their morphogenesis, genome structures, rep-
Plant rhabdoviruses are transmitted by vectors such as lication at intracellular sites, and vector species [4, 5]. These
aphids, planthoppers, leafhoppers, mites, and fungi [1–3]. viruses, except for varicosa- and dichorhaviruses, have a
Plant rhabdoviruses are taxonomically classified in six negative-sense single-stranded RNA genome of 11–15 kb
genera: Alphanucleorhabdovirus, Betanucleorhabdovirus, encapsulated in a bullet-shaped or bacilliform particle [3,
4]. The genome of betanucleorhabdoviruses encodes at least
six proteins: nucleoprotein (N), phosphoprotein (P), puta-
Handling Editor: Ralf Georg Dietzgen.
tive movement protein (P3), matrix protein (M), glycopro-
* Su‑Heon Lee tein (G), and viral RNA-dependent RNA polymerase (RdRP;
suheon@knu.ac.kr L), in the order 3′-N–P–P3–M–G–L–5′, with inverted com-
* Jae Sun Moon plementary termini [2–4].
jsmoon@kribb.re.k Cnidium officinale (Korean name: chunkug) is a peren-
1 nial plant belonging to the family Umbelliferae that is used
Biosystem and Bioengineering Program, University
of Science and Technology (UST), Daejeon 34141, as a side dish and as a medicinal plant to treat a variety of
Republic of Korea diseases in Asia, particularly in Korea and China [8]. Several
2
Plant System Engineering Research Center, Korean Research viruses have been reported to infect C. officinale, including
Institute of Bioscience and Biotechnology, Daejeon 34141, cnidium vein yellowing virus (CnVYV-1 and CnVYV-2;
Republic of Korea family Secoviridae) [9], cnidium virus X (family Alphaflexi-
3
Plant Biotechnology Research Division, Amhara Agricultural viridae) [10], and cycas necrotic stunt virus (family Secoviri-
Research Institute, Bahir Dar, Ethiopia dae) [11]. In this study, the complete genomic sequence of
4
Department of Biology, School of Mathematics and Natural a betanucleorhabdovirus, tentatively named “cnidium virus
Sciences, Mongolian National University of Education, 1” (CnV1), identified in a C. officinale plant, is described
Ulaanbaatar, Mongolia for the first time.
5
School of Applied Bioscience, College of Agriculture In May 2018, C. officinale plants that showed virus-like
and Life Sciences, Kyungpook National University, symptoms (leaf deformation, mild mottling, severe stunting,
Daegu 98411, Republic of Korea

13
Vol.:(0123456789)

974 M. T. Belete et al.

leaf mosaic, and vein clearing) were collected in the garden and RevertAid Reverse Transcriptase (Thermo Scientific,
of the National Institute of Forest Science in Seoul, South Waltham, MA, USA) [12]. The cDNA was amplified by
Korea. In total, 43 samples were individually crushed to a PCR using AccuPower® ProFi Taq PCR PreMix (Bioneer,
fine powder, and equal aliquots (3 g) from each leaf were Daejeon, South Korea) with 12 pairs of primers (Supple-
pooled for high-throughput sequencing (HTS). The total mentary Table S1). The purified RT-PCR products were
RNA was extracted from the pooled sample using an easy- ligated directly into the RBC T&A Cloning Vector (RBC
spin™ Total RNA Extraction Kit (iNtRON Biotechnology, Bioscience, Taipei, Taiwan) and used to transform compe-
Sangdaewon-dong, South Korea). Ribosomal RNA was tent Escherichia coli (DH5α) cells. The inserts from at least
eliminated using a Ribo-Zero™ rRNA Removal Kit (plant three positive clones containing each RT-PCR product were
leaf) (Epicenter, Madison, WI, USA), and a cDNA library extracted using a HiGene™ Plasmid Mini Prep Kit (version
was generated using an Illumina TruSeq™ RNA Sample 2.0) and sequenced by Macrogen.
Prep Kit (Illumina, San Diego, CA, USA). DNA sequencing To determine the 5’- and 3’-terminal sequences, we
was performed on an Illumina HiSeq 4000 platform at Mac- used a 5’and 3’ RACE system for completing the genome
rogen (Seoul, South Korea). A total number of 722,575,410 sequence of CnV1. Total RNA was extracted from a C. offic-
paired-end reads and 72.9 Gbp of total reads with a read inale sample using a WizPrep™ Plant RNA Mini Kit, and
length of 101 bp were obtained. The resulting raw reads this RNA was used as the template for 5’ and 3’ RACE pro-
were filtered to eliminate low-quality reads and adapter tocols. To identify the 3’-terminal sequences, poly(A) tails
sequences. After trimming, the total number of reads was were added to the 3′-terminal ends using a Poly(A) Polymer-
707,516,224 and total number of bases was 71.1 Gbp. The ase Tailing Kit (Epicenter) and SuperScript III (version 2.0;
high-quality reads were assembled de novo into transcript Invitrogen, Carlsbad, CA, USA) for first-strand cDNA syn-
contig sequences using the Trinity program (https://​github.​ thesis and amplification using a poly(T) primer with gene-
com/​trini​tyrna​seq/​trini​tyrna​seq/​wiki). A total of 218,239 specific primers (Supplementary Table S1). To complete
assembled transcripts ranging from 201 to 16,841 nt were the sequence of the 5’ end, the Invitrogen 5’ RACE system
used to query the virus RefSeq database. (version 2.0) was used. The sequence at each end of the
The assembled contigs were subjected to a BLASTn amplicon was determined from at least five clones in both
search of the National Center for Biotechnology Informa- directions. The DNAMAN program (version 5.2.10; Lynnon
tion GenBank database, which showed that one large contig BioSoft, QC, Canada) was used to assemble and compile all
of 13,994 nucleotides (nt) corresponded to a novel nucle- of the overlapping sequence fragments. Once completed and
orhabdovirus that shared 66.7% nt sequence identity with corrected, the full 14,002-nt genomic sequence of CnV1 was
sowthistle yellow vein virus (SYVV, GenBank accession deposited in the GenBank database under accession number
no. MT185675) and green Sichuan pepper nucleorhabdovi- MZ983390.
rus (GSPNuV, GenBank accession no. MH323437), and In an analysis of the genome structure of CnV1, six
68% identity with black currant-associated rhabdovirus open reading frames (ORFs) were predicted in the 14,002-
(BCaRV, GenBank accession no. MF543022) and carda- nt sequence, in the order 3′-nucleocapsid protein (N, 465
mom vein clearing virus (CdVCV, GenBank accession no. amino acids, 51.6 kDa); phosphoprotein (P, 333 amino acids,
MN273311). In addition, virus-like contigs corresponding 36.5 kDa); putative cell-to-cell movement protein (P3, 323
to cnidium vein yellowing virus 1, cnidium vein yellowing amino acids, 36.2 kDa); matrix protein (M, 269 amino acids,
virus 2, cycas necrotic stunt virus, and cucumber mosaic 30.1 kDa); glycoprotein (G, 650 amino acids, 74.3 kDa);
virus were also detected. and large (L; RdRP, 2118 amino acids, 240.3 kDa)-5′. The
To identify the nucleorhabdovirus and determine its full- CnV1 sequence has negative polarity. A 228-nt 3’ leader
length genome sequence, primers were synthesized to pro- (l) sequence precedes the N gene, and a 192-nt 5’ trailer (t)
duce a set of 12 partially overlapping reverse transcription sequence follows the L gene. Therefore, the genome organ-
(RT)-PCR products that covered most of the CnV1 genome, ization of CnV1 is 3′-l–N–P–P3–M–G–L–t-5′ (Fig. 1B).
except its extremities (Supplementary Table S1). On May CnV1 has the highest amino acid (aa) sequence identity
5, 2021, another C. officinale plant with similar symptoms in P3, M, and L—38.6, 35.7, and 52%, respectively—to
(Fig. 1A) was collected from Yeongyang-dun and used as the respective viral proteins of BCaRV (Supplementary
a template for RT-PCR. All of the primer pairs designed Table S2) [7]. The CnV1 genes are separated by conserved
from the HTS contig effectively amplified fragments of the intergenic regions, which include the preceding gene’s poly-
expected sizes. The pairwise nt sequence identity between adenylation signal, an untranslated intergenic spacer, and
the genome sequence obtained by HTS and the second sam- the transcription start of the following gene (Supplementary
ple sequenced from RT-PCR amplicons was 99.5%. RNA Table S3) [2, 4].
was extracted using a WizPrep™ Plant RNA Mini Kit, and Nineteen of the 22 terminal nucleotides of the 3'
cDNA was synthesized using the primer N25 (25-mer) leader sequence and the 24 terminal nucleotides of the 5'

13
Complete genome sequence of cnidium virus 1 975

(A)

(B) 465 aa 333 aa 323 aa 269 aa 650 aa 2118 aa

3’ l N P P3 M G L t 5’
192 nt 228 nt

Coting
1 3 5 7 9 11 5’RACE
3’RACE 2 4 6 8 10 12

(C)
ORF Gene Size I Putative gene function Predicted NLS cNLS mapper score II Predicted nuclear export site III
1 N 465 aa Nucleocapsid Protein 433RAAKRKVPEP442 5.6 I352, L354
2 P 333 aa Phosphoprotein Bipartite 4.3 ND
3 P3 323 aa Movement protein Bipartite 4.6 L57,S58,L59,G60,A61,L62,E63,M64
4 M 269 aa Matrix protein 215LRRIIKKKRHL225 7.4 ND
5 G 650 aa Glycoprotein Bipartite 3.9 L23, L594, L597
6 L 2118 aa Polymerase 1646DKGIKRKRGNR1656 8 I242, L359, L362,L878,L882,

I. nt=nucleotides; aa=amino acid residues

II. cNLS Mapper score: predicted using cNLS Mapper
III. Nuclear export signals: predicted by NetNES 1.1 Server, analysis and prediction of leucine rich nuclear export signals ND: no detection, I:
Isoleucine, L: leucine, S: serine, G: glycine, A: alanine, E: glutamic acid, M: methionine.

Fig. 1  A Image of the Cnidium officinale plant in which cnidium and the locations of the 3′ leader (l) and 5′ trailer (t) are indicated at
virus 1 was detected, showing virus-like symptoms. B Schematic the ends. C cNLS Mapper scores. A cNLS score of 8, 9, or 10 implies
illustration of the CnV1 genome organization and the relative posi- that the protein is exclusively localized to the nucleus; a score of 7 or
tions of the contig and RT-PCR and RACE PCR product. Positions 8 indicates partial nuclear localization; a score of 3, 4, or 5 indicates
of ORFs encoding proteins (N, P, P3, M, G, and L; via positive sense) both nuclear and cytoplasmic localization; and a score of 1 or 2 indi-
are shown, and the lengths of the amino acid (aa) sequences are indi- cates cytoplasmic localization.
cated. The intergenic regions are indicated by inverted red triangles,

trailer sequence (5'-GAG​ACA​GAA​ACA​CaCCA​TAA​ac… CnV1 clustered with the betanucleorhabdoviruses and was
agUUA​UGG​aGUG​UUU​CUG​UCU​Cua-3', presented as the most closely related to BCaRV and CdVCV (Fig. 2), which
antigenome strand) are complementary, potentially form- were also the most similar based on pairwise sequence
ing a panhandle structure in which the 5′ trailer has three identity values (Supplementary Table S2) and genome
unmatched overhanging nucleotides (Fig. 1B) [6, 7]. To organization (Fig. 1B). The ORFs predicted in the viral
investigate the taxonomic position of the virus, a phylo- genome were analyzed using SnapGene Viewer. cNLS
genetic tree was constructed by the maximum-likelihood Mapper [7] was used to predict nuclear localization sig-
method and the Jones–Taylor–Thornton (JTT) matrix nals (NLSs), showing that CnV1 proteins contain a clas-
model in MEGA X [2] with 500 bootstrap replicates, based sical monopartite or bipartite NLS. Based on their cNLS
on aa sequences of the L protein (Fig. 2). In this analysis, scores, the L protein is expected to be exclusively nuclear,

13

976 M. T. Belete et al.

Betanucleorhabdovirus

Dichorhavirus

Alphanucleorhabdovirus

Gammanucleorhabdovirus

Cytorhabdovirus

Vesiculovirus

Fig. 2  Maximum-likelihood phylogenetic tree created in MEGA X at the nodes but are only shown if the value is above 50%. CnV1 is
using the WAG + G + I + F model, based on aa sequences of the indicated by a red diamond. The accession numbers of viruses used to
polymerase (l-protein). Bootstrap values for 500 replicates are given construct the phylogenetic tree are shown in brackets

whereas the M protein is predicted to have partial nuclear The ICTV species demarcation criteria stipulate that
localization, and the N, P, P3, and G proteins are predicted viruses belonging to new species within the genus Beta-
to have both nuclear and cytoplasmic localization. Leu- nucleorhabdovirus should share less than 75% complete
cine-rich nuclear export signals (NESs) were predicted in nucleotide sequence identity [1–4]. The pairwise percent
the N, P3, G, and L proteins using the NetNES 1.1 server identity values calculated from a multiple sequence align-
[4, 9] (Fig. 1C). ment constructed using ClustalW2.1 [12] revealed that the
The negative-sense, single-stranded RNA genome of complete nucleotide sequences of CnV1 and other beta-
rhabdoviruses acts as a template for a series of polyade- nucleorhabdoviruses share sequence identity in the range
nylated mRNA transcripts that correspond to the individual of 37.8%–56% (Supplementary Table S2). Thus, CnV1
ORFs and the untranslated leader RNA [2]. On the positive should be classified as a member of a new species in the
(antigenome) strand of CnV1, the conserved gene-junction genus. The maximum-likelihood phylogenetic analysis of
sequences located between the ORFs contain a putative CnV1 shows that it is most closely related to the betanu-
polyadenylation/transcript termination signal (5’-AUA​UAA​ cleorhabdoviruses (Fig. 2).
GAA​AAA​-3’), a putative untranscribed dinucleotide spacer The genomic sequence obtained in this study  will
(5’-CC-3’), and a putative transcript initiation motif (5’- help in the further characterization of this virus and the
AAC-3’). These intergenic iterons are 100% identical to the identification of potential alternative hosts and insect
iterons of BCaRV and other related viruses (Supplementary vectors.
Table S4) [3, 4].

13
Complete genome sequence of cnidium virus 1 977

Supplementary Information  The online version contains supplemen- 4. Bejerman N, Dietzgen RG, Debat H (2021) Illuminating the plant
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00705-0​ 21-0​ 5348-9. rhabdovirus landscape through metatranscriptomics data. Viruses
13(7):1–25
Acknowledgements  This work was supported by IPET (Korea Insti- 5. Dietzgen RG, Bejerman NE, Goodin MM, Higgins CM, Huot
tute of Planning and Evaluation for Technology in Food, Agriculture, OB, Kondo H, Martin KM, Whitfield AE (2020) Diversity and
Forestry and Fisheries; Project No. AGC1762111), Ministry of Agri- epidemiology of plant rhabdoviruses. Virus Res 281:197942
culture, Food and Rural Affairs, Republic of Korea. We thank Edanz 6. Dietzgen RG, Innes DJ, Bejerman N (2015) Complete genome
(www.​edanz.​com/​ac) for editing a draft of this manuscript. sequence and intracellular protein localization of Datura yellow
vein nucleorhabdovirus. Virus Res 205:7–11
7. Wu LP, Yang T, Liu HW, Postman J, Li R (2018) Molecular char-
Declarations  acterization of a novel rhabdovirus infecting blackcurrant identi-
fied by high-throughput sequencing. Adv Virol 163(5):1363–1366
Conflict of interest  All authors declare no conflict of interest. 8. Choi HS, Kim MSL, Sawamura M (2002) Constituents of the
essential oil of Cnidium officinale Makino, a Korean medicinal
Ethical approval  This article does not contain any studies with animals plant. Flavour Fragr J 17(1):49–53
or human participants performed by any of the authors. 9. Yoo RH, Zhao F, Lim S, Igori D, Kim SM, An TJ, Lee SH, Moon
JS (2015) The complete genome sequences of two isolates of cnid-
ium vein yellowing virus, a tentative new member of the family
Secoviridae. Adv Virol 160(11):2911–2914
References 10. Honma H, Tsushima D, Kawakami H, Fujihara N, Tsusaka T,
Kawashimo M, Nishimura T, Fuji S (2019) Complete nucleotide
sequence of a new potexvirus, ‘Cnidium virus X’, isolated from
1. Walker PJ, Breyta R, Blasdell KR, Calisher CH, Dietzgen RG,
Cnidium officinale in Japan. Adv Virol 164(7):1931–1935
Fooks AR, Fieitas-astúa J, Kondo H, Kurath G, Kuzmin IV,
11. Igori D, Lee HK, Yang HJ, Lee DS, Kim SY, Kwon B et al (2020)
Longdon B, Stone DM, Tesh RB, Tordo N, Vasilakis N, Anna
First report of the cycas necrotic stunt virus infecting Cnidium
E, Walker PJ (2020) Virus taxonomy the ICTV report on virus
officinale in South Korea. Plant Dis 104(12):3275–3275
classification and taxon nomenclature rhabdoviridae chapter
12. Belete MT, Gudeta WF, Kim SE, Igori D, Moon JS (2021) Com-
rhabdoviridae vertebrate host vertebrate host, arthropod vector,
plete genome sequence of Codonopsis torradovirus A, a novel
pp 1–84
torradovirus infecting Codonopsis lanceolata in South Korea. Adv
2. Stenger DC, Burbank LP, Wang R, Stewart AA, Mathias C, Goo-
Virol 0123456789:1–4
din MM (2020) Lost and found: rediscovery and genomic charac-
terization of sowthistle yellow vein virus after a 30+ year hiatus.
Publisher's Note Springer Nature remains neutral with regard to
Virus Res 284:197987
jurisdictional claims in published maps and institutional affiliations.
3. Walker PJ, Blasdell KR, Calisher CH, Dietzgen RG, Kondo H,
Kurath G, Longdon B, Stone DM, Tesh RB, Tordo N, Vasilakis N,
Whitfield AE (2018) ICTV virus taxonomy profile: Rhabdoviri-
dae. J Gen Virol 99(4):447–448

13