Plant viruses in the genus Pelarspovirus (family Tombusviridae) are non-enveloped, 28-34 nm in diameter, have capsids with T = 3 icosahedral symmetry, and possess a positive-sense RNA genome approximately 3.8-3.9 kb in length. All members of the family Tombusviridae, with the exception of members of the genus Dianthovirus, have a non-segmented genome that is neither 3'-polyadenylated nor 5'-capped [4]. Viruses belonging to the genus Pelarspovirus exhibit a number of distinctive characteristics. To translate their five gene products, they use five distinct reading frames [5]. The five ORFs encode two replication-supporting proteins, two movement proteins (MP), and a capsid protein (CP) [5]. One subgenomic RNA contains the genetic information for both movement proteins and the capsid protein [3]. In addition, some members of the genus Pelarspovirus have a non-canonical start codon (GUG or CUG) that initiates the expression of the ORF coding for MP2 [5]. The absence of an AUG codon in any frame between the AUG initiation codon of MP1 and the CP open reading frame is another typical feature of pelarspoviruses [5].

Quercus aliena Blume (family Fagaceae), commonly known as Oriental white oak, is native to eastern Asia and is a major deciduous tree in Korean natural forests [2]. The acorns of this species, which is widespread in eastern Asia, contribute to basic food chains in forest ecosystems [1]. The starchy nuts of mature trees may also help to mitigate long-term climate change through oak tree carbon sequestration [6].

In 2021, oak trees exhibiting virus-like symptoms, such as leaf rolling, leaf dwarfing, and browning of new leaves were observed at an elevation of 1,150 m at Changjuk-dong, Taebaek-si, and Gangwon-do, South Korea. Symptomatic leaves were collected, ground in the laboratory using liquid nitrogen, and stored at -80 °C. A WizPrep Plant RNA Mini Kit (Wizbiosolutions, Seongnam, South Korea) was used to isolate total RNA. Ribosomal RNA was depleted from total RNA using a Ribo-Zero rRNA Removal Kit (Plant Leaf) according to the manufacturer's instructions (Epicentre, Madison, WI, USA). Next, a cDNA library was produced using a TruSeq RNA Sample Prep Kit (Illumina San Diego, CA, USA) and sequenced on an Illumina Novaseq 6000 platform (Illumina) by Macrogen (Seoul, South Korea). The resulting raw reads were filtered using the Trimmomatic program (http://www.usadellab.org/cms/?page=trimmomatic) to yield 595,758,264 high-quality reads. Trimmed high-quality reads were assembled de novo into contig sequences using the Trinity program, and each contig was newly annotated through a BLASTx search of the NCBI GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Among the identified plant virus contigs, one contig comprising 4,023 nucleotides (nt) was similar to pelarspovirus sequences available in the database. In particular, it shared 69.92% amino acid sequence identity (query coverage: 55%) with clematis chlorotic mottle virus (ClCMV, GenBank accession no. AYI50027). The sequence of the contig corresponding to the nearly full-length genome of the virus was confirmed by RT-PCR. For this, we designed four specific primer sets overlapping by more than 100 bp, based on the 4,023-nt contig sequence and made a new total RNA extract using a WizPrep Plant RNA Mini Kit (Supplementary Table S1). Reverse transcription (RT)-PCR performed using the four specific primer sets and SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, South Korea) yielded bands only of the expected sizes from oak. All amplicons were purified using an AccuPrepPCR Purification Kit (Bioneer, Daejeon, Korea) and cloned, and at least three clones for each RT-PCR product were sequenced by Macrogen (Seoul, South Korea). CLC Main Workbench v.21 (Insilicogen, Yongin, South Korea) was used to assemble the resultant sequences. Because pelarspoviruses do not have a poly(A) tail at the 3′ end, total RNA was adenylated using a Poly(A) Polymerase Tailing Kit (Epicentre, Madison, WI, USA) before amplification. cDNA was synthesized from total RNA using a RevertAid RT Reverse Transcription Kit (Thermo Scientific, Waltham, MA, USA) and random N25 primers, and PCR products were obtained from known sequences using AccuPower ProFi Taq PCR PreMix (Bioneer). Next, 5′-terminal sequences were generated with the aid of a 5′-rapid amplification of cDNA ends (5′-RACE) kit (Invitrogen, Waltham, MA, USA). All amplicons were also cloned into a TA cloning vector, and at least six clones were sequenced in both directions by Macrogen. The sequences were then assembled using CLC Main Workbench v.21, revealing that the newly identified virus has a RNA genome size of 3,940 nt. This virus was named “quercus leafroll virus” (QLRV), and its genome sequence was deposited in the GenBank database under accession number OR193176.

The 3,940-nt-long genome sequence of QLRV contains five ORFs and lacks a poly-A tail (Fig. 1). Pairwise comparisons of QLRV RNA sequence regions with those of viruses belonging to 36 Tombusviridae species, using CLUSTAL W 2.1 [9], revealed 17.9-44.2% nt sequence identity (Supplementary Table S2). The putative p27 (27-kDa) protein encoded by the first ORF, which is 702 nt (234 codons) long, shares the highest amino acid sequence identity, 52%, with the p27 protein of clematis chlorotic mottle virus (ClCMV). ORF1 at nt positions 703-705 has an amber stop codon, UAG, that is likely read through to produce a 87-kDa replicase protein (ORF2). The sequence 5′-AAA UAG GGA-3′ (nt 700-708) in QLRV, which includes the amber stop codon, is a readthrough sequence matching the pattern (A/C/U)(A/U)A UAG(G/C)(G/A/U) [7]. The ORF2-encoded protein is the most similar to those of ClCMV and Rosa rugosa leaf distortion virus (RrLDV), with 67% and 51% aa sequence identity, respectively. ORF3 (nt 2254-2475,) encodes a movement protein (MP1) of 73 aa that is 61% identical to the MP1 protein of pelargonium chlorotic ring pattern virus (PCRPV). The current criteria for species demarcation in the genus Pelarspovirus are less than 75% amino acid sequence identity in the RdRp and CP to other members of the genus [5]. Since the similarity between QLRV and other investigated pelarspoviruses is below the thresholds for both RdRp and CP, we conclude that QLRV is a representative of a new species in the genus Pelarspovirus.

Fig. 1
figure 1

Schematic representation of the genome organization of quercus leafroll virus (QLRV). ORFs 1, 2 and 4 are located in frame 2, ORF 3 is located in frame 1, and ORF 5 is located in frame 3. The arrow between ORF 1 and 2 indicates the readthrough site at an amber stop codon

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To analyze the relationship of QLRV to other members of family Tombusviridae, we compared RdRp and CP amino acid sequences using MEGA 11 [8]. In the deduced phylogenetic trees shown in Fig. 2, QLRV is grouped with other members of the genus Pelarspovirus, again strongly suggesting that QLRV is a new member to the genus Pelarspovirus in the family Tombusviridae. This is the first report of a pelarspovirus infecting Q. aliena in South Korea.

Fig. 2
figure 2

Phylogenetic trees based on RdRp (A) and capsid protein (B) amino acid sequences of quercus leafroll virus (QLRV) and members of the family Tombusviridae. The trees were constructed by the maximum-likelihood method in MEGA 11, with 1000 bootstrap iterations. Numerical values indicate bootstrap percentage values. The accession numbers of the amino acid sequences of the proteins used to construct the tree are shown in parentheses

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