Elm Blunervirus 1: A Novel Hexapartite Blunervirus Infecting Ulmus parvifolia in China

Abstract

The genus Blunervirus comprises plant viruses that infect a diverse range of plants, but no blunervirus has been reported infecting elm trees (Ulmus parvifolia) in China to date. Using high-throughput sequencing and reverse-transcription PCR assays, a novel blunervirus, tentatively named elm blunervirus 1 (ElmBlV1), was identified from a symptomatic elm plant (Ulmus parvifolia) in China. The genome of ElmBlV1 harbors canonical molecular features of blunerviruses and comprises six RNA segments (RNAs1–6), with RNA5 and 6 being two additional genomic components not reported in known blunerviruses. Sequence analyses revealed amino acid (aa) identity of ElmBlV1 proteins ranging from 25.9% (polyprotein encoded by RNA1) to 64.2% (movement protein encoded by RNA4) relative to reported blunerviruses and include five orphan open reading frames. Phylogenetically, ElmBlV1 is most closely related to blueberry necrotic ring blotch virus. Furthermore, ElmBlV1 P37 localizes to both plasmodesmata and the nucleus. Additionally, the RNA reads mapping revealed high read coverage was observed on RNAs3–4 for this virus. To our knowledge, this is the first report of a blunervirus infecting an elm tree in China. Our results enrich the diversity of known viruses in the genus of Blunervirus and expand our understanding of their genomic characteristics and molecular biology.

1. Introduction

Elm tree (Ulmus spp.), a plant species in the family Ulmaceae, is one of the most common and valuable amenity trees and is widely cultivated in cities, urban spaces and rural areas for its tolerance to adverse environmental conditions and high survival [1,2]. For a long time, Dutch elm disease (DED), a vascular wilt disease caused by fungi of Ophiostoma spp., has been considered a devastating threat to elm plants health [3,4,5]. In addition to fungal pathogens, elm trees are known to host seven viruses, including a tombusvirus (tomato bushy stunt virus, TBSV), a carlavirus (elm carlavirus, ElmCV), two ilarviruses (elm mottle virus, EMoV; elm mosaic virus, EMV), and three nepoviruses (cherry leaf roll virus, CLRV; tobacco ringspot virus, TRSV; tomato ringspots virus, ToRSV) [6,7,8,9,10]. Notably, most of these viral infections have been documented in European countries, while no elm-infecting viruses have been identified in China to date.

The genus Blunervirus belongs to the family Kitaviridae (order Martellivirales), which was formally established by the International Committee on Taxonomy of Viruses (ICTV) to encompass plant-infecting RNA viruses with multipartite genomic RNAs and evolutionary links to arthropod-infecting negeviruses [11,12,13,14]. To date, the ICTV recognized only three species in genus of Blunervirus: Blunervirus vaccinii, Blunervirus camelliae, and Blunervirus [12,15,16,17]. These viruses are new emerging pathogens of economically important horticultural plants (blueberry, tea and tomato) and some of the blunerviruses are transmitted by phytophagous mites [12,18,19,20]. Classically, blunerviruses were defined by a quadripartite positive-sense single-stranded RNA (+ssRNA) genome, with each segment encoding at least one open reading frame (ORF). RNA1 and RNA2 encode replication-associated polyproteins harboring domains of methyltransferase (Met), helicase (Hel), and RNA-dependent RNA polymerase (RdRp); RNA3 encodes the structural virion membrane protein (SP24, conserved across Kitaviridae); and RNA4 encodes the movement protein (MP, 30 K superfamily) [16,17,21]. One of the species demarcation criteria for blunervirus is >25% aa sequence divergence in the polyprotein encoded by RNA1 [13,15].

In 2021, virus-like symptoms of leaf mosaic and chlorosis were observed on an elm tree grown in Urumqi city, Xinjiang Uygur Autonomous Region, China. Subsequently, high-throughput sequencing (HTS) of the leaf tissue identified a novel blunervirus infecting the plant. Notably, four genomic RNA segments (RNAs1–4) of the virus had been previously amplified and deposited in the NCBI database in 2022. Building on foundational work, this study completes the genomic characterizations of the virus, designated as elm blunervirus 1 (ElmBlV1), by experimentally validating two additional RNA segments (RNA5 and RNA6). Furthermore, we provided the first experimental evidence for the subcellular localization of a blunervirus MP.

2. Materials and Methods

2.1. Plant Material

In April 2020, an elm plant (Ulmus parvifolia) (ID: YS) with viral-like symptoms of mosaic and chlorotic spots (Figure 1) was observed along a street in Urumqi City, Xinjiang Uygur Autonomous Region of China. The leaf samples were collected and used for further HTS analysis and subsequent gene amplification.

Figure 1.

Virus-like symptoms on leaves of an elm plant (Ulmus parvifolia) used for high-throughput sequencing (HTS).

2.2. High Throughput Sequencing and Bio-Information Analysis

For RNA-seq, total RNA was extracted from elm leaf tissue using the Trizol (Invitrogen) according to the manufacturer’s instructions. Ribosomal RNA (rRNA) was depleted using the Epicentre Ribo-ZeroTM rRNA removal kit (Epicentre, Madison, WI, USA). Then, a cDNA library was constructed with a TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA). Finally, the RNA-seq was carried out on an Illumina HiSeq XTen sequencer (Biomarker Biology Technology Ltd. Company, Beijing, China), generating 150 nt paired-end reads.

The raw sequencing data from the Illumina platform were processed to trim adaptor sequences and filtered for low-quality reads using FASTP (version 1.5.6) [22]. Then, the trimmed reads were de novo assembled into contigs using Velvet (version 1.2.08) and IDBA-UD (version 1.1.1) [23,24] with k-mer values set to 80, 90 and 110, respectively. Finally, the resulting contigs were screened for sequence identities against the NCBI databases (http://www.ncbi.nlm.nih.gov/ accessed on 20 January 2022) using BlastX and BlastN programs.

To identify additional genomic RNA segments of ElmBlV1, we employed the UTR-backed iterative BLASTn (UTR-iBLASTn) method recently developed by Zhang et al. [25]. Briefly, the method began by constructing a reference database comprising conserved 3’UTR sequences of ElmBlV1 previously identified RNAs1–4. The assembled contigs derived from HTS clean data were then subjected to iterative BLASTn searches against this database with e-value of 1 × 10⁻². The putative viral contigs were trimmed of redundant coding regions, and their UTRs were integrated to expand the database iteratively. This cycle continues until no new UTR-conserved contigs are identified, ensuring capture of all potential genomic segments.

2.3. Amplification of Viral Genome RNAs

In order to obtain the complete genome sequence of the virus, 13 specific primer pairs were designed based on the assembled contigs sequence (Table S1). Total RNA was extracted from leaf tissue using a cetyltrimethylammonium bromide (CTAB) method [26]. About 500 ng of total RNA was used to generate cDNA with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA) and random primer pd(N)6 (TaKaRa, Dalian, China). The reverse transcription mixture was incubated at 37 ℃ for 1.5 h. The 5’termini of ElmBlV1 genomic RNAs were amplified using a commercial SMARTer RACE kit (TaKaRa, Dalian, China) following the manufacturer’s protocol. Briefly, for 5’ RACE, the first-strand cDNA was synthesized using the 5’-CDS Primer A and SMART II A oligonucleotide (provided in the kit) with reverse transcriptase, followed by PCR amplification with gene-specific primers and Universal Primer A Mix (UPM) (Table S1). The 3’ RACE-ready cDNA was generated using the oligo(dT) anchor primer M4-T, and PCR was performed with gene-specific primers and M4 (Table S1). The RT-PCR solutions and conditions were similar to those described previously [27,28], except that annealing temperature and extension time varied depending on the primer sets used in each reaction and the lengths of the amplicons. All the PCR products were gel-purified and ligated into the pMD18-T vector (TaKaRa, Dalian, China). At least three positive clones of each amplicon were sequenced at Shanghai Sangon Biological Engineering and Technology and Service Co. Ltd., Shanghai, China.

2.4. RT-PCR Detection of the ElmBlV1

During 2021–2025, leaf samples were collected from 43 individual elm trees. A total of 30 samples were collected from elm trees grown in Xinjiang Uygur Autonomous Region, 5 samples were collected from Henan Province, and 8 samples were collected from elm trees in Hubei Province. The presence of ElmBlV1 was confirmed by simultaneous RT-PCR amplifications using the four sets of specific primer, CPF/CPR, which were used to amplify a 407 bp segments targeting the gene of SP24 domain in RNA3, 5F/5R and 6F/6R (Table S1), which were used to amplify a 536 and 579 bp fragments including coding region of RNA5 and RNA6, respectively.

2.5. Sequence Analyses

The overlapped sequences were assembled using Vector NTI software version 11.5 (Invitrogen, Carlsbad, CA, USA). The ORFs were predicted by ORFfinder tool (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 10 August 2025). Conserved domains of viral proteins were determined utilizing CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/docs/cdd_search.html, accessed on 1 September 2025) [18], interPro (https://www.ebi.ac.uk/interpro/, accessed on 1 September 2025) [18], and SMART tools (http://smart.embl-heidelberg.de. accessed on 1 September 2025) [19]. Transmembrane helices (TMHs) were predicted by TMHMM tool (http://www.cbs.dtu.dk/services/TMHMM, accessed on 5 September 2025). The WG/GW argonaute-binding domains were predicted using the Agos online server (http://www.combio.pl/agos, accessed on 5 September 2025). The potential signal peptide sites were predicted using SignalP (versiton:6.0; http://www.cbs.dtu.dk/services/SignalP-6.0/, accessed on 5 September 2025). The aa Asequence identities were computed using BlastP program in the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 7 December 2025). The secondary structure prediction of P37 of ElmBlV1 was performed using software JPred4 (http://www.compbio.dundee.ac.uk/jpred4/, accessed on 10 September 2025). The phylogenetic tree was constructed using the maximum-likelihood (ML) method with the recommended best-fit model LG+G and 1000 bootstrap replicates in MEGA 12 software [29].

2.6. Subcellular Localization Analysis

To evaluate the subcellular localization of a putative MP (P37) of ElmBlV1, ORF4a (missing stop codon) was cloned into the N-terminus of eYFP-pCNF3 by using XbaI and BamHI digestion sites. The sequence-validated construct was transformed into Agrobacterium tumefaciens strain GV3101 (Weidi, Shanghai, China). To visualize intracellular structures, the plasmids encoding mCherry-CMV3a (plasmodesma marker) [30] and mCherry-H2B (nuclear marker) [31] were co-infiltrated with the P37-eYFP construct into Nicotiana benthamiana leaves via agrobacterium-mediated infiltration, respectively, following the protocol described previously [30]. The fluorescence signals of reconstitution in infiltrated leaf section were acquired using confocal laser scanning microscopy (CLSM; Leica Microsystems, TCS-SP8, Wetzlar, Germany) with an HC PL APO CS2 63×/1.20 WATER objective.

3. Results

3.1. Identification of a Novel Blunervirus Infecting Elm by HTS

In total, HTS generated 66,314,198 (98.8% of raw data) clean reads from the sequencing library. Among the assembled contigs: four contigs (IDs: contig-100_9985; contig-100_1834; contig-100_182 and contig-100_119) with the length of 1665 bp–6190 bp sharedaa sequence identities ranging from 25.9% to 64.2% to proteins of two replication-associated polyproteins, SP24 and MP, encoded by RNAs1–4 of viruses in the genus Blunervirus. The result indicated the presence of a novel blunervirus infecting elm plant, which was provisionally named “elm blunervirus 1” (ElmBlV1). Total RNA was extracted from the HTS sample, and a virus-specific primer pair JC-F/JC-R (Table S1) was employed to confirm the presence of ElmBlV1 in HTS sample. Cloning and Sanger sequencing revealed that the yielded 574 bp amplicon shared 99.8% sequence identity with the corresponding region of contig-100_119 at nt level. In addition, no other viral contig was detected in the HTS dataset.

The complete 5’ and 3’ terminal sequences of all six genomic RNA segments of ElmBlV1 were experimentally confirmed by RACE assays, and the full-length genomic sequences were assembled by RT-PCR and RACE sequencing results. The genome of ElmBlV1 is composed of six RNA segments (GenBank accession numbers: OL865294–OL865297, and PX905634–PX905635). The viral RNA1 was 5868 nt in length and possessed a single ORF (nt: 54–5651) encoding 210.2 kDa replication-associated polyprotein (P210) of 1865 amino acids (aa) (Figure 2). This putative protein P210 contained three conserved domains of methyltransferase (Met; pfam01660; Interval: 133–372; E-value: 5.13 × 10⁻⁸), ovarian tumor (OTU; cd22792; Interval: 660–789; E-value: 4.19 × 10⁻¹⁴), and viral_helicase1 (Hel; pfam01443; Interval: 1522–1826; E-value: 6.58 × 10⁻¹⁹). No TMH and SP were predicted in the P210 of ElmBlV1. BlastP analysis revealed that ElmBlV1 P210 shared the highest aa sequence identity of 25.9% with homologous protein encoding by wheat blunervirus 1 (WhBlV1) (Table 1), a potential novel blunervirus identified through data-driven virus discovery (DDVD) approach.

Figure 2.

Genome organization and the profile distribution of RNA-seq reads along six RNA segments of elm blunervirus 1 (ElmBlV1). Proteins encoded by RNAs1-6 of ElmBlV1 are shown as green boxes with the protein name. An approximate size scale is shown on the top of genomic RNA segments.

Table 1.

BlastP and bioinformatics analyses of proteins encoded by elm blunervirus 1 (ElmBlV1).

RNA Segment	ORF	Size (nt)	Protein	BlastP hit (a)	Cover (%)	Identity (%)	Domain (b)
RNA1	ORF1	5598	P210	WhBlV1	97.0	25.9	Met, OTU, Hel
RNA2	ORF2	3450	P132	PiBlV1	85.0	39.6	Hel, RdRp
RNA3	ORF3 a	372	P13	-	-	-	-
	ORF3 b	780	P29	-	-	-	-
	ORF3 c	585	P22	BNRBV	73.0	36.6	SP24, GW
	ORF3 d	405	P14	BNRBV	41.0	50.0	-
RNA4	ORF4 a	1014	P4 a	BNRBV		64.2	3A
	ORF4 b	252	P9	-	-	-	-
RNA5	ORF5	1848	P47	-	-	-	GW
RNA6	ORF6	1086	P41	-	-	-

(a): WhBlV1, wheat blunervirus 1; PiBlV1, pine blunervirus 1; BNRBV, blueberry necrotic ring blotch virus. (b): Met, methyltransferase; OTU, ovarian tumor; Hel, helicase; RdRp, RNA-dependent RNA polymerase; 3A, 3A movement protein; SP24, structural protein of 24 kDa; GW, glycine-tryptophan (GW) dipeptide. -, no hit.

The viral RNA2 had 3960 nt with an ORF (nt: 251–3700) that encoded a 1149 aa polyprotein with predicted molecular weight (MW) of 131.9 kDa (P132) (Figure 2). It exhibited the highest aa sequence identity of 39.6% with the corresponding polyprotein of pine blunervirus 1 (PiBV1) (Table 1). In silico analysis identified two conserved domains within ElmBlV1 P132 protein: a Hel (pfam01443; Interval: 225–466; E-value: 2.23 × 10⁻¹⁹) localized at the N terminus, a kitaviridae-specific RNA-dependent RNA polymerase (RdRp; cd23254; Interval: 820–1083; E-value: 1.90 × 10⁻¹⁰⁷) positioned at the C terminus (Table 1).

The viral RNA3 was 2966 nt and harbored four ORFs (Figure 2). From 5’ to 3’end, the first ORF encoded for a 123 aa protein with a MW of 13 kDa (P13). The second ORF encoded for a 259 aa protein (P29). A TMH (aa: 221–240) and an SP with a predicted cleavage site at Ala_23/Gly₂₄ were identified in the P29 of ElmBlV1. The third ORF encoded a 193 aa protein (P22), a homolog of SP24 of blunerviruses (pfam16504; Interval: 25–160; E-value: 2.57 × 10⁻¹⁸). Four TMHs (aa: 35–57, 77–99, 106–128 and 143–165) were predicted within the ElmBlV1 P22 protein. It shared the highest aa sequence identity of 35.7% with SP24 homolog of blueberry necrotic ring blotch virus (BNRBV). The 3’-terminal ORF encoded a 134 aa protein (P14) orthologous (50% aa identity) to a hypothetical protein encoded on RNA3 of BNRBV, and the presence of an SP with a predicted cleavage Trp₂₄/Gln₂₅. The proteins P13 and P29 of ElmBlV1 showed no similarity to any protein in the current NCBI databases by BlastP search (Table 1).

The viral RNA4 was 2073 nt in size and possessed two ORFs. From the 5’ termini, the large ORF (nt: 410–1423) putatively encoded for a 337 aa protein with weight of 37.4 kDa (P37) (Figure 2). It shared the highest aa sequence identity of 64.2% to the MP of BNRBV (Table 1) and contained a conserved 3A MP domain (pfam00803; Interval: 49–233; E-value: 1.62 × 10⁻²⁵), supporting its role in cell-to-cell movement. Multiple aa sequence alignments based on both sequence and secondary structure prediction were performed using software JPred4. The result showed that the secondary structure pattern of ElmBlV1 P37 shared a similarity to that previously reported for MPs of the viruses belonging to the 30 K superfamily and also possessed an aspartic acid (D₁₇₁) of 30 K-specific LxD/N50–70G (Figure S1). Additionally, a glycine-tryptophan (GW) dipeptide motif (aa: 41–56; p-value: 9.57 × 10⁻¹), conserved among viral RNA silencing suppressors, was identified at the N-terminus of ElmBlV1 P37. In the 3’-terminal ORF (nt: 1763–2014) on RNA4, an 83 aa protein with MW of 9.3 kDa (P9) was encoded. A TMH (aa: 57–79) and an SP with a predicted cleavage site at Thr50/Arg51 were identified in ElmBlV1 P9, which showed no similarity to known proteins in current public databases (Table 1).

The viral RNA5 was 1848 nt and contained a single ORF of 1245 nt (nt: 206–1450) and produced a polypeptide of 47.4 kDa (P47). The RNA6 was 1698 nt and harbored an ORF (nt: 374–1459) encoding a 361 aa protein with a predicted MW of 41.2 kDa (P41) (Figure 2). Neither ElmBlV1 protein P47 nor P41 showed significant similarity to known proteins via BlastP searches (Table 1). Intriguingly, a GW dipeptide motif (aa: 232–252; p-value: 9.18 × 10⁻¹) was identified in the central region of ElmBlV1 P47.

Sequence analysis revealed that the 5′and 3′untranslated regions (UTRs) of ElmBlV1 RNA segments ranged from 83 to 439 nt and 85 to 650 nt, respectively, and each RNA segment exhibited high AU-richness, 59–77% at 5′UTR and 57–67% at 3′UTR. Notably, a highly conserved stretch of 8 nt (5′-AUUAGUU-3′) was observed at 3’ ends of each RNA segment (Figure S2), and this terminal sequence was unambiguously confirmed by RACE assays.

3.2. Phylogenetic Analysis

To clarify the phylogenetic relationship of ElmBlV1 within genus Blunervirus, phylogenetic trees were constructed using aa sequences of the protein RdRp and SP24, together with homologous proteins from previously characterized blunerviruses and selected members of the family Kitaviridae. The results revealed that ElmBlV1 clustered closely with BNRBV to form a subclade within the Blunervirus clade with a high bootstrap support (>95%) and distinctly separated from the TPA-elm blunervirus (a virus assembled from public data) (Figure 3). Consistently, the phylogenetic tree constructed based on the SP24 protein sequences exhibited a similar topological structure (Figure S3).

Figure 3.

Maximum-likelihood (ML) phylogenetic tree constructed based on amino acid (aa) sequences of the RdRp domain of elm blunervirus 1 (ElmBlV1) and selected members of the family Kitaviridae. ElmBlV1 is highlighted with a red solid dot. The ML tree was constructed using MEGA 12 software under the LG+G substitution model with 1000 bootstrap replicates. Members of the genus Blunervirus are circled in blue. Experimentally validated tentative blunerviruses are circled in cyan, while those identified solely via the data-driven virus discovery (DDVD) approach are circled in yellow. The referred sequences were marked by their virus names followed by GenBank accession numbers. The number of RNA segments for each blunervirus is shown in the right panel.

3.3. P37 Encoding by RNA4 of ElmBlV1 Localized at PD

Given that protein ElmBlV1 P37 encoded by RNA4 possessed a 3A MP domain, we predicted that it was involved in viral cell-to-cell movement, and the subcellular localization of viral P37 in epidermal cells of N. benthamiana leaves was examined by confocal laser scanning microscopy. It was found that the fluorescence signals of P37-eYFP were observed as punctate spots along the cell wall and co-localized with the plasmodesma (PD) marker protein CMV-3a-mCherry at PDs in the epidermal cells (Figure 4A). In addition to PD localization, P37-eYFP was also accumulated in the nucleus and formed aggregates of variable sizes in the cytoplasm (Figure 4B).

Figure 4.

Subcellular localization assays of elm blunervirus 1 (ElmBlV1) P37 encoded by ORF4a in RNA4 in epidermal cells of Nicotiana benthamiana leaves. The fusion proteins CMV3a-mCherry and H2B-mCherry were used as plasmodesmata (PD) (A) and nucleus (B) markers, respectively. The aggregated bodies formed by proteins ElmBlV1-P37 are denoted by arrows. Punctate colocalization of ElmBlV1-P37 with CMV3a-mCherry at PDs is denoted by arrowheads. Enlarged section of each image is highlighted with box. Images were acquired at 2 days after agroinfiltration under a confocal microscope at 63×/1.20 WATER objective. Scale bar = 20 μm.

3.4. Distribution of ElmBlV1-Derived RNA Reads Across the Viral Genome

After obtaining the complete ElmBlV1 genomic RNA sequence, virus-derived RNA reads from the HTS dataset were mapped to the six genomic segments. A total of 238,377 (0.04% of total clean reads) clean reads aligned to the six genomic RNA segments of ElmBlV1, covering nearly the entire length of all RNA segments. The RNA3 and RNA4 exhibited higher RPKM values compared to the other four genomic RNA segments (Table S2), with prominent read coverage peaks observed in their mapping profiles (Figure 2).

3.5. Virus Detection

To investigate the occurrence of ElmBlV1 plants, during 2021–2025, leaf samples were collected from a total of 43 individual elm plants, including 30 from the Xinjiang Uygur Autonomous Region, 5 from Henan Province and 8 from Hubei Province. A total of 5 of 43 samples exhibited symptoms of chlorotic spots and/or leaf mosaic. The RT-PCR assays were performed for virus detection, employing the three sets of primer pairs CPF/CPR, 5F/5R and 6F/6R for simultaneous amplification. The results showed that all of the tested samples were negative for the ElmBlV1, indicating that this novel blunervirus currently has a local distribution in elm plant, and has not spread widely in the investigated regions.

4. Discussion

Since the discovered blunervirus, BNRBV, was first characterized in 2013, only three species had been recognized in the genus of Blunervirus over a decade year [12,15,16,17]. With the rapid advancement and widespread application of HTS, the identification of novel viruses has been greatly facilitated, and viral species diversity including 18 tentative blunerviruses has also significantly expanded [25,32]. In the present study, we identified and molecularly characterized a novel blunervirus, ElmBlV1, from a symptomatic Ulmus pumila plant in China, and determined its complete genomic sequence by combining HTS, RT-PCR and RACE assays. Our work provides the first definitive evidence of a novel blunervirus naturally infecting elm in China, expanding understanding of the molecular characteristics and host range of viruses in the genus of Blunervirus.

ElmBlV1 possessed a unique hexapartite genome (RNAs1–6), expanding the ICTV-approved quadripartite core genome (RNAs1–4) with two previously unreported accessory segments (RNA5–RNA6), which is the highest number of genomic segments reported in blunervirus to date. Similar to BNRBV, TPNRBV isolate Ca1 (TPNRBV-Ca1), Ailanthus blunervirus 1 (AiBV1), apple blunervirus 1 (ApBV1), Paulownia tomentosa-TSA blunervirus (PTBV) [16,17,25], ElmBlV-1 harbors highly conserved sequences in the 3′ UTRs of all six genomic segments, and high AU-richness in UTRs further validate RNAs5–6 as genomic components. Conserved terminal UTR sequences serve as natural markers of viral genomic components and signatures of evolutionary conservation and are hypothesized to ensure the synchronized transcription and expression of both core and accessory genomic segments [12,18,25,33]. Leveraging this conserved structural feature, the RNA5 of TPNRBV-Ca1, -AiBV1 -ApBV1 [25], as well as RNA5 and RNA6 of ElmBlV1, were identified by UTR-based iterative BLASTn. Furthermore, this also leaves the possibility that additional genomic RNA segments may be discovered for members of the genus Blunervirus in subsequent investigations. In contrast, no conserved 3′UTR sequences have been detected in tomato fruit blotch virus (ToFBV) [15]. Coincidentally, a terminal conservation is also observed in viruses of the genus Emaravirus. RNA5 and RNA6 of fig mosaic virus (FMV) and RNA5 of Actinidia chlorotic ringspot-associated virus (AcCRaV) were precisely identified based on the terminal features [28,34]. Regarding poly(A) tails, ElmBlV1, TPNRBV, and ToFBV have poly(A) tails at the 3′ ends of their RNA segments, while BNRBV lacks this feature [12,15,16,17].

Like previously characterized blunervirus, ElmBlV1 encoded two replication-associated domains across different proteins: P210 encoded by RNA1 harbored Met and Hel domains, P132 encoded by RNA2 contained Hel and RdRp domains. This domain arrangement mirrors the blunervirus’ canonical replication module distribution, while the RdRp domain has also been found in proteins encoded from RNA5 of ApBV1 and RNA3 of AiBV1 [25], respectively. Furthermore, RNA1 of both AiBV1 and nanmu blunervirus 1 (NaBlV1) exhibited a peculiar arrangement of Met-Hel-RdRp domain architecture, which was not observed in other blunerviruses, suggesting a potential evolutionary divergence in replication module organization within the genus of Blunervirus [25,32].

RNA3 exhibited variable ORF numbers, with counts ranging from 2 to 5 among the characterized blunerviruses [12,32]. ElmBlV1 RNA3 contained four ORFs, a genomic feature shared with BNRBV, AiBV1-RNA4, and nine other blunerviruses [17,25,32]. This was distinct from ToFBV, PTBV, NaBlV1 and podocarpus celatus blunervirus 1 (PcBlV1), which each harbored five ORFs in RNA3 segment [15,25,32]. The third ORF of ElmBlV1 RNA3 encoded P22, an ortholog of the SP24 protein that represents the most conserved molecular hallmark within the genus Blunervirus [12,14,21,35]. The protein translated from the second ORF in ElmBlV1 RNA3 contained an SP and a TMH, which is analogous to that of BNRBV and ToFBV [12,15,17].

A single ORF encoding MP was predicted on RNA4 of BNRBV, TPNRBV, and 11 other tentative blunerviruses [15,16,17,25,32]. ElmBlV1 RNA4 differed from reported blunerviruses that consisted of two ORFs. The first ORF that encoded the putative P37 containing conserved 3A motif was located in the 5′ region of ElmBlV1 RNA4; in contrast, the counterpart of ToFBV was located in the 3′ region of RNA4 [15]. Furthermore, subcellular localization assays demonstrated that the ElmBlV1 P37 localized to PD, sharing a similar pattern to other plant viral MPs in 30 K superfamiy [36,37,38], supporting that P37 is the MP of the ElmBlV1. In addition, P37 also localizes in the nucleus, and is different from that of citrus leprosis virus C (genus Cileviruses, family Kitaviridae) MP, which exhibited PD and endoplasmic reticulum (ER) localizations [39,40]. Remarkably, the ElmBlV1 P37 contained a conserved “GW” motif, which is essential for RNA silencing suppressor (RSS) activity in plant viruses [41,42], suggesting that P37 may have a dual role in viral movement and host RNA silencing defense. The nuclear accumulation of ElmBlV1 P37 further supported these dual functions, as nuclear localization was often critical for RSS proteins that interfere with host silencing pathways [43,44]. Likewise, the protein P3 of Barley yellow striate mosaic virus (a cytorhabdovirus) and MP of citrus tatter leaf virus (a capillovirus) also exhibited dual functions in mediating both cell-to-cell movement and RSS activity [45,46]. In contrast, recent studies have not detected MPs in 10 tentative blunerviruses [25,32], suggesting variability in viral movement strategies within the genus.

In general, the genomes of kitaviruses harbored orphan ORFs (ORFans), a feature that reflects the viral evolutionary strategy of acquiring novel genes to facilitate adaptation to diverse plant hosts [12,25,47,48]. ElmBlV1 encoded six ORFan proteins with no homologs in public databases. Intriguingly, protein P47 (encoded from RNA5) of ElmBlV1 contained a “GW” motif, which hints at a potential role in evading host immune defenses [42]. Similarly, an ORFan protein P68 of PTBV with a predicted NLS homologous to that of the cucumoviral 2b protein, a known RSS, was speculated to confer RSS activity [21,43]. This parallels findings in viruses in the genus of Emaravirus, where non-core proteins (beyond P1–P4) were linked to similar functions [49,50,51,52,53,54].

Analysis of the genomic RNA distribution profile revealed high coverage depth across the viral RNA3 and RNA4. Our study provided the first report on RNA reads distribution profile for a blunervirus. SP24, a major structural protein of viral particles, encoded by RNA3, the expression level was consistent with the critical role in viral packaging. Besides assisting the transport of viral ribonucleoprotein (vRNP) [39], P37 encoded by RNA4 of ElmBlV1 was predicted to participate in RNA silencing suppression. This prediction aligns with observations in RSS genes of other plant viruses, including P23 of Citrus tristeza virus (genus: Closterovirus) and P5 of pear chlorotic leaf spot-associated virus (genus: Emaravirus) [52,53].

According to the recently approved new species demarcation for the genus Blunervirus, less than 75% aa sequence identify in the polyprotein encoded by RNA1 is required for new species [11,15]. In fact, the polyprotein encoded by RNA1 of ElmBlV1 showed the highest aa identities of 25.6% with the homologous protein of WhBlV1 and was below the threshold of species demarcation. Thus, we concluded ElmBlV1 to be a novel virus in the genus Blunervirus. Recently, four contigs of a TPA-elm blunvervirus assembled from public American Ulmus americana transcriptome data were recorded in GenBank by Sudharshan Reddy et al. [32]. Although the assembled contig sequences lack experimental validation, these findings raised the possibility that elm trees may be hosts for at least two blunerviruses. Similarly, hibiscus (Hibiscus spp.), woody ornamental plants, are natural hosts for two distinct kitaviruses: hibiscus green spot virus 2 and hibiscus yellow blotch virus [55,56]. The rare detection of ElmBlV1 in the tested samples indicated an extremely low positive detection rate, suggesting limited viral dissemination and making it difficult to determine an association between the virus and the observed symptoms.

5. Conclusions

In conclusion, we have determined the complete genomic sequence of ElmBlV1, which harbors the canonical molecular features of blunerviruses and comprises two additional genomic RNAs (RNAs5–6). It exhibits low sequence identities with all previously characterized blunerviruses and is closely related to BNRBV. Our study presents the first experimental evidence that a blunervirus MP localizes to PD and characterizes the distribution pattern of blunervirus-derived RNA reads. Nevertheless, its symptomatology associated with virus infection and the functions of its ORFans remain undefined. Thus, further research is needed to clarify the biological properties and protein functions of ElmBlV1.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/v18020266/s1, Figure S1. Alignment of amino acid sequences of elm blunervirus 1 (ElmBlV1) P37 and selected 30 K superfamily movement proteins (MP) of two cytorhabdoviruses (LNYV and ADV) and two tobamoviruses (TMV and TVCV) using the MAFFT(A), and the conserved D₁₇₁ of ElmBlV1 was in the downstream of β-stands (yellow) (B). GenBank accession numbers of sequences used for the analysis are as follows: TMV, tobacco mosaic virus (NC_001367); TVCV, turnip vein-clearing virus (U03387); ADV, alfalfa dwarf virus (KP205452); LNYV, lettuce necrotic yellows virus (AJ867584). Figure S2. Multiple alignment of nucleotide sequences at the 3′ un-translation region (3′UTR) of six RNA segments of elm blunervirus 1 (ElmBlV1). The conserved sequences at 3′ UTR of each RNA were highlighted with a red box. Figure S3. Maximum-likelihood (ML) phylogenetic tree constructed based on amino acid (aa) sequences of the SP24 domain of elm blunervirus 1 (ElmBlV1) and selected members of the family Kitaviridae. ElmBlV1 is highlighted with a red solid dot. The ML tree was constructed using MEGA 12 software under the LG+G+F+I substitution model with 1000 bootstrap replicates. The referred sequences were marked by their virus names followed by GenBank accession numbers. Table S1. The primers used for the genome amplification and RT-PCR detection of elm blunervirus 1 (ElmBlV1). Table S2. Mapping analysis of the reads matched genomic RNAs of elm blunervirus 1 (ElmBlV1).

Author Contributions

Conceptualization, Y.W. (Yanxiang Wang), L.Z., Y.W. (Ying Wang) and J.B.; Methodology, L.Z., J.L., K.Y. and Y.W. (Ying Wang); Software, Y.W. (Yanxiang Wang), L.Z. and J.X.; Investigation, L.Z., J.X., W.C., J.L., K.Y., X.S., J.T., X.X. and J.B.; Data curation, Y.W. (Yanxiang Wang) and J.B.; Writing—original draft, Y.W. (Yanxiang Wang) and Y.W. (Ying Wang); Writing—review and editing, Y.W. (Ying Wang) and J.B.; Funding acquisition, Y.W. (Yanxiang Wang), Y.W. (Ying Wang) and J.B. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The genomic sequences of elm blunervirus 1 are available under GenBank accession No.: OL865294–OL865297, PX905634–PX905635.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This study was financially supported by the Youth Project of Natural Science Foundation of Hubei Province (Grant No. 2023AFB405), the Key Project of the Scientific Research Program of Hubei Provincial Department of Education (Grant No. Q20242507), the National Natural Science Foundation of China (Grant No. 32402394), the Central Government Guided Local Science and Technology Development Project of Hubei Province (Grant No. 2023EGA041), and Special Forestry Science and Technology Program of the National Forestry and Grassland Administration of China: General Survey of Pathogenic Microorganisms Associated with Invasive Alien Plants in Forest, Grassland and Wetland Ecosystems of Xinjiang Uygur Autonomous Region (Grant No. 65000022P00789110010X).