,
1
,
1
,
2
,
2
and
1,
*
Sung-Woong Kim
1
Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University, Gwangju 61185,
Korea
Hyo-Jeong Lee
1
Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University, Gwangju 61185,
Korea
Sena Choi
2
Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365,
Korea
In-Sook Cho
2
Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365,
Korea
Rae-Dong Jeong
1
Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University, Gwangju 61185,
1
Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University, Gwangju 61185,
Korea
2
Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365,
Korea
Corresponding author.
*
Total readsMinimum read length (nt)Maximum read length (nt)Mean read length (nt)Total readsMinimum read length (nt)Maximum read length (nt)Mean read length (nt)
Iksora cococinea
434,106896,990317.1349,0692090344.6SRR23561814
Table 2
List of identified JaVH reads in
Ixora coccinea
sample
De novo
assembly of MinION reads was performed with genome sequences of JaVH isolate (JaVH-CNU) (
). A total of 796 reads (0.22% of the total reads) were mapped with 95.5% nucleotide coverage and 95.1% nucleotide identity. To obtain the complete genome sequence of JaVH, the consensus sequences were finally mapped against a reference viral genome (NC055545; at 60× coverage depth and at least 20% of support fraction for base-call ambiguity). In addition, Sanger sequencing after reverse transcription polymerase chain reaction (RT-PCR) was performed for filling in any sequence gaps using virus-specific primers (Fwd: 5′-TCC AAG GCG AAT GCT CTC TCT GT-3′/Rev: 5′-GGA TTG TAG TGG AGC GGT GAA AAA CCC-3′), which amplify the fragments between the aligned reads, designed based on the consensus sequence. The RT-PCR reaction mixture (final volume 20 μl) contained 10 μl SuPrimerScript RT Premix (Genet Bio, Daejeon, Korea), 2 μl RNA template, 2 μl of each of the 10 μM forward and reverse primers, and diethylpyrocarbonate-treated water. Amplification reaction was conducted under the following conditions: 50°C for 30 min; 95°C for 5 min; 35 cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 30 s; and 72°C for 5 min. The 5′ and 3′ end sequences of JaVH were determined by rapid amplification of cDNA end (RACE) technique using a SMARTer RACE 5′/3′ Kit (Clontech, Mountain View, CA, USA). The complete genome consensus sequence of the JaVH infecting
I. coccinea
was deposited in GenBank (
{"type":"entrez-nucleotide","attrs":{"text":"LC757512","term_id":"2451843290","term_text":"LC757512"}}
LC757512
).
The genome structure of JaVH-CNU was predicted using the ORFfinder software (
http://www.ncbi.nlm.nih.gov/orffinder
). The RNA genome of JaVH-CNU consisted of 3,867 nucleotides (nt), with a total of four open reading frames (ORFs) (
). The 5′ and 3′ untranslated regions were found to be 19 nt and 226 nt in length, respectively. The first ORF (ORF1) comprised 729 nt and has potential to read-through a downstream ORF, resulting in production of a 2,301 nt sequence encoding the RNA-dependent RNA polymerase (RdRP). The ORF2 (189 nt) and ORF3 (252 nt) encoded movement protein. The ORF4 (1020 nt) encoded a coat protein (CP) (
).
To confirm the presence of JaVH in
I. coccinea
, RT-PCR was performed using JaVH-specific diagnostic primer sets (
Dey et al., 2018
), and the RT-PCR product (657 bp) was sequenced by Sanger sequencing (
). The obtained sequences confirmed the presence of JaVH (data not shown).
The complete genome of JaVH-CNU exhibited sequence identities ranging from 88.4% (JaVH-Hunan,
{"type":"entrez-nucleotide","attrs":{"text":"MH231179","term_id":"1554431107","term_text":"MH231179"}}
MH231179
) to 90.3% (JaVH-Fujian, NC055545) with other JaVH isolates. In addition, to compare the amino acid sequences, the complete amino acid sequences of RdRP and CP of JaVH-CNU were aligned with those of other JaVH isolates available on GenBank using BioEdit software version 7.2.5 (Ibis Biosciences, Carlsbad, CA, USA). Pairwise distances were calculated using the PASC algorithm (NCBI, Bethesda, MD, USA) available on the GenBank database. JaVH-CNU shared amino acid sequence identities of 94.9–95.3% in RdRP (
) and 94.4–96.2% in CP (
).
To assess the phylogenetic relationship among JaVH-CNU and other JaVH isolates, the complete amino acid sequences of the RdRP and CP of JaVH-CNU, along with those of other JaVH isolates, were used in phylogenetic analysis. The phylogenetic tree was constructed using the MEGA 10.0 tool (
Kumar et al., 2018
). The alignments were used to infer neighbor-joining trees in MEGA 10.0 with Tamura-Nei model and 1,000 bootstrap replicates as described by
Lee et al. (2022)
. Phylogenetic analysis of RdRP and CP revealed that the JaVH-CNU differed from the existing isolates in GenBank. The RdRP and CP of JaVH-CNU were clustered separately with different JaVH isolates, probably having different biological characteristics (
).
The spread of plan viruses through global trade is a significant concern. The international movement of infected plant materials or their products can introduce viruses to new geographic locations and host plant species, potentially leading to the emergence of new disease. Moreover, the movement of insect vectors, such as aphids or whiteflies, can also facilitate the spread of plant viruses through global (
Amari et al., 2021
). To successfully respond to an unexpected outbreak of plant viruses, it has become increasingly important to establish rapid and reliable diagnostic methods for effective disease management.
With the advantages of nanopore sequencing, this study sequenced and identified the full genome of a JaVH isolate from
I. coccinea
, and it took only 48 h from RNA extraction to virus identification in the laboratory, providing rapid diagnosis for disease survey and management. The JaVH, which has been recently characterized from
Jasminum sambac
(
Zhuo et al., 2017
) and
J. multiflorum
(
Dey et al., 2018
), belongs to a member of the genus
Pelarspovirus
in the family
Tombusviridae
. Despite the JaVH isolates reported in other countries and hosts, the genome of JaVH seems to be highly variable based on phylogenetic analyses of complete amino acid sequences of RdRP and CP (
Dey et al., 2018
). It is worth noting that this is first report of a natural infection of JaVH in
I. coccinea
worldwide. Thus, it is urgently required to survey the incidence of JaVH in
I. coccinea
in Korea, and more importantly, to evaluate the disease risk in different types of crops and ecologies.
Acknowledgments
This work was carried out with the support of Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ014947032023) Rural Development Administration, Republic of Korea.
Footnotes
Conflicts of Interest
No potential conflict of interest relevant to this article was reported.
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