First report of Raphanus sativus cryptic virus 2 on cabbage seeds (Brassica rapa subsp. pekinensis) imported from Italy

Abstract

Raphanus sativus cryptic virus 2 (RsCV-2), an unclassified species belonging to the family Partitiviridae, was identified in cabbage seeds (Brassica rapa subsp. pekinensis) imported from Italy using high-throughput paired-end RNA sequencing. Three contigs derived from RsCV-2 isolate PQ shared nucleotide sequence homologies (approximately 94, 87, and 86%) with dsRNA1, 2, and 3 of RsCV-2, respectively. Phylogenetic analysis and pairwise comparison showed that RsCV-2 isolates were more closely related to deltapartitiviruses. To the best of our knowledge, this is the first report of RsCV-2 on B. rapa subsp. pekinensis in the world.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13337-021-00744-w.

Raphanus sativus cryptic virus 2 (RsCV-2), which is an unclassified species belonging to the family Partitiviridae, was first identified from Raphanus sativus in China in 2006 [1]. Since then, no further RsCV-2 sequences have been available in NCBI GenBank. A large number of members of the family Partitiviridae have two segmented double-stranded RNA genomes [2, 4], but some species including RsCV-2 have more than three genomic RNAs. The host range of viral members of the family Partitiviridae generally includes fungi, plants and protozoans [2, 4].

To identify virus populations in cabbage seeds (Brassica rapa subsp. pekinensis) imported from Italy, high-throughput sequencing (HTS) was performed with a pooled sample of the cabbage seeds. The sample was previously identified to be infected with turnip yellow mosaic virus (TYMV) using reverse transcription (RT) polymerase chain reaction (PCR). TYMV has been designated as one of the plant quarantine viruses in Korea. Total RNA was extracted from the finely ground pooled sample using STEADi 24 system (GeneAll, Seoul, Korea) with Automated Plant Seed Viral RNA Kit (InnogeneTech, Gimpo, Korea). Removal of ribosomal RNA and construction of cDNA library were conducted using TruSeq® Stranded Total RNA Library Preparation Kit with Ribo-Zero™ Plant (Illumina, San Diego, CA, USA). Library construction, high-throughput paired-end RNA sequencing using the Illumina HiSeq4000 system, and analysis of about 6.7 Gbp of the raw data (101 bp of paired-end reads; and a total of 66,497,718 reads) were performed at Macrogen, Inc. (Daejeon, Korea). The HTS result reconfirmed the existence of TYMV in cabbage seeds.

Interestingly, three contigs derived from RsCV-2, which were 1718-nt (9207 reads), 1506-nt (5559 reads), and 1443-nt (8202 reads) in length, respectively, were also obtained (Supplementary Fig. S1). The NCBI BLASTn searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) of the contigs showed that they shared nucleotide sequence homologies (approximately 94, 87, and 86%) with dsRNA1, 2, and 3 of RsCV-2, respectively. Three open reading frames (ORF) were searched in three contig sequences corresponding to three genomic dsRNAs of RsCV-2 using CLC Main Workbench 6 (QIAGEN, Germantown, MD, USA), and a conserved domain/motif (replicase) was identified in the dsRNA1 using the Pfam (protein families database: http://pfam.xfam.org/; [3]) analysis (Supplementary Fig. S1). To reconfirm the existence of the virus, three primer sets (RsCV-2_RNA1, 5′-TTT CAC CGT ACC AGC GCG AA-3′ and 5′-TGT AAT AGT RCA CGC AGC AC-3′; RsCV-2_RNA2, 5′-TCG CCG CTA ACC AAG CAC AT-3′ and 5′-TCC CGT TYA GAC AAG ATT TGG-3′; and RsCV-2_RNA3, 5′ - GTA CAC ACA CTA ACC AAC TTG-3′ and 5′- TGC AAA GAC ACA CCA CTT GG-3′) were designed using the three contigs and reference genome sequences (NC_010343, NC_010344, and NC_010345). RT-PCR was performed using SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, Korea). Each primer set amplifies full length of each ORF, and the three amplicons were sequenced using Sanger method (Supplementary Fig. S1). Consensus sequences of RT-PCR amplicons obtained from RsCV-2 isolate PQ identified in cabbage seeds were deposited in the NCBI GenBank (accession nos. MZ437436, MZ437437, and MZ437438).

Phylogenetic analysis and pairwise comparison were conducted with complete nucleotide sequences of ORFs of dsRNA1 and 2 using CLC Main Workbench 6. The results showed that two RsCV-2 isolates were more closely related to deltapartitiviruses (Fig. 1 and Supplementary Fig. S2). Comparative analysis (BLAST searches, phylogenetic trees, and pairwise comparisons) of three sequences determined from HTS and RT-PCR showed that the virus present in cabbage seeds is RsCV-2 isolate PQ. To the best of our knowledge, this is the first report of RsCV-2 on B. rapa subsp. pekinensis in the world. Various further studies are needed to elucidate the characteristics of RsCV-2 such as pathogenicity and seed transmission.

Fig. 1.

Phylogenetic analysis and pairwise comparison of dsRNA1 of RsCV-2 isolate PQ. A Phylogenetic analysis was performed based on an alignment using coding nucleotide sequences of dsRNA1 of some species belonging to the family Partitiviridae. Tree was constructed by the maximum likelihood phylogeny method using CLC Main Workbench 6. B Pairwise comparison was also performed based on the alignment. All reference genome sequences were obtained from NCBI database (https://www.ncbi.nlm.nih.gov/genome/viruses/)

Supplementary Information

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Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not describe any studies with human participants or animals performed by any of the authors.