Identification, molecular characterization and phylogenetic analysis of a novel nucleorhabdovirus infecting Paris polyphylla var. yunnanensis

A novel betanucleorhabdovirus infecting Paris polyphylla var. yunnanensis, tentatively named Paris yunnanensis rhabdovirus 1 (PyRV1), was recently identified in Yunnan Province, China. The infected plants showed vein clearing and leaf crinkle at early stage of infection, followed by leaf yellowing and necrosis. Enveloped bacilliform particles were observed using electron microscopy. The virus was mechanically transmissible to Nicotiana bethamiana and N. glutinosa. The complete genome of PyRV1 consists of 13,509 nucleotides, the organization of which was typical of rhabdoviruses, containing six open reading frames encoding proteins N–P–P3–M–G–L on the anti-sense strand, separated by conserved intergenic regions and flanked by complementary 3′-leader and 5′-trailer sequences. The genome of PyRV1 shared highest nucleotide sequence identity (55.1%) with Sonchus yellow net virus (SYNV), and the N, P, P3, M, G, and L proteins showed 56.9%, 37.2%, 38.4%, 41.8%, 56.7%, and 49.4% amino acid sequence identities with respective proteins of SYNV, suggesting RyRV1 belongs to a new species of the genus Betanucleorhabdovirus.

www.nature.com/scientificreports/ and Varicosavirus) based on the replication sites and morphogenesis in the cytoplasm or nucleus of infected cells, and their genome architecture [14][15][16] . Betanucleorhabdovirus genomes encode 6 canonical proteins in the conserved order of 3′-to-5′: nucleoprotein (N), phosphoprotein (P), movement protein (P3), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The genome lacks additional accessory open reading frames (ORFs). The genes are flanked by conserved junction regions 3′-AUU CUU UUU GG UUG-5′ separating their genes, and complementary 3′-leader and 5′-trailer sequences 15,17,18 . Recently, with the application of High Throughput Sequencing (HTS), more and more plant rhabdoviruses were discovered 19,20 . The previously reported betanucleorhabdoviruses infect dicot and monocot plants and several of them are transmitted by aphids in which they also replicate 21 .
In this study, we used the HTS platforms and RT-PCR to determine the complete genome of a new betanucleorhabdovirus infecting P. polyphylla var. yunnanensis, provisionally named Pairs yunnanensis rhabdovirus 1 (PyRV1) based on analysis of genomic organization, sequence homology and phylogeny.

Methods
Virus source. A field survey was conducted to find out medicinal plant diseases in Yunnan province, China in 2019. During the survey, virus infected Paris polyphylla var. yunnanensis plant samples were collected from the medicinal plant plantation in QuJing city of YunNan province. The diseased leaf material was homogenized at ratio 1:5 (w/v) in 0.1 mol/L sodium phosphate Buffer, pH7.0, with carborundum powder as an abrasive agent. Then it was mechanically smeared with the pestle onto the young leaves of Nicotiana benthamiana, N. glutinosa, N. tabacum var. K326 and Sonchus oleraceus and injected with a medicinal injector into the leaf vein of P. polyphylla var. yunnanensis to determine if the virus was sap-transmitted.
Experimental research on plant samples, including the supply of plant material, complies with institutional, national and international guidelines and legislation.
Negative stain and electron microscopy observation. For electron microscopy, 0.1 g symptomatic leaves from P. polyphylla var. yunnanensis were directly cut in 20 µL 2.5% isoamyl alcohol until the tissues was completely homogenized. A pioloform carbon-coated copper grid was floated for 3.5 min on the crude sap to absorb the viral particles. The grids were then transferred onto one drop of 20 µL negative staining Buffer (2% ammonium molybdate at pH 6.5) and stained for 2 min. Finally, the grids were dried by absorbing moisture with filter paper. The virions were examined under a transmission electron microscope (FEI TECNAI G2, Ther-moFisher Scientific, Hillsboro, Oregon, USA).
High throughput sequencing and read assembly. Total RNA was extracted from the diseased P. polyphylla var. yunnanensis leaves using TRIzol (Ambion, Hillsboro, Oregon, USA) following the manufacturer's protocol. Ribosomal RNA was depleted using a RiboZero kit (Illumina, San Diego, CA, USA) according to the manufacturer's protocol. Next, a cDNA library was synthesized using the TruSeq Stranded Total RNA with Ribo-Zero Gold (Illumina). The cDNA library obtained was subjected to deep sequencing using the Illumina HiSeq 2500.
Raw data (raw reads) were processed using Trimmomatic 22 . Low quality reads were removed. Next, the clean reads were assembled into expressed sequence tag clusters (contigs) and de novo assembled into transcripts by using Trinity (version: 2.4) with the paired-end method 23 . The longest transcript was chosen as a unigene based on the similarity and length of a sequence for subsequent analysis. The assembled-contigs (above 150 nt) were subjected to local BLAST searches against the reference viral sequence (RefSeq) database of NCBI (the E-value cut-off was > 0.05 for local tBlastx).

RT-PCR amplification of virus genomes.
First, the partial fragment (Amplicon 2) of the rhabdovirus L gene containing a conserved polymerase motif were amplified using the previously reported degenerate primers RhabFor (GGATMTGGGGBCATCC) and RhabRev (GTCCABCCY TTT TGYC) 24 . In addition, specific primers were designed based on the contig sequences obtained above to amplify and verify the genome sequence (Table S1). Total RNA was extracted using the TRIzol Reagent (ThermoFisher Scientific) from the diseased leaves. The RT-PCR reactions were performed using the Prime ScriptTM One Step RT-PCR Kit (TaKaRa, Shiga, Japan). The amplification conditions were as follows: 50 °C for 30 min, 94 °C for 3 min, 30 cycles of 94 °C for 30 s, 48 °C for 30 s and 72 °C for 1 min, 72 °C for 10 min, 4 °C for 10 min. The resulting PCR amplicons were resolved in 1% agarose gels, stained with Ts-GelRed (TsingKe, Beijing, China), and cloned into the pMDTM19-T Vector Cloning Kit (TaKaRa). Three positive recombinant plasmids of every fragment were sequenced in bothdirections.
Genomic 5′ and 3′-termini were determined using SMARTer 5′/3′ RACE kits (TaKaRa, Shiga, Japan). For the 5′-terminus, first-strand cDNA was produced with random priming according to the user manual. Rapid amplification of 5′-end was performed with the primer GPS-5′end: GAT TAC GCC AAG CTT GGG AAG CCC ATA TGT GAC CCG AAGAC (identical to 13,146-13,171 of the genomic 5′-end). A band of approximately 700 bases was obtained. For the 3′-terminus, total RNA was added with a poly(A) tail using Poly(A) Polymerase (TaKaRa), then used as a template to synthesize the first-strand cDNA of the 3′-end. Rapid amplification of the 3′-end was performed with the primer GPS-3end: GAT TAC GCC AAG CTT TGG AGG AGA GGA GAA CAC ATT CCC TCC (reverse complementary with 478-504 of genomic 3′-end). A band of approximately 730 bases was obtained. The PCR products of both ends were cleaned and cloned according to the manufacturer's instructions. Eight colonies were sequenced in both directions. RT-PCR detection of PyRV1 from field samples. Based on the alignments of the nucleotide sequences encoding conserved polymerase motifs in betanucleorhabdoviruses L genes, the primers specific to PyRV1 were designed (8307F:5′-TGG AGG ATA TGG GGT CAC CCGAT-3′, 9046R: 5′-TCA GAC ATG GTG ATC ATC GGG AAA TA-3′) to detect field samples. RT-PCR was conducted using the Prime ScriptTM One Step RT-PCR Kit (TaKaRa, Shiga, Japan) according to the above-mentioned protocol. Ten plant samples with virus-like symptoms were randomly collected from plantation of Paris polyphylla var. yunnanensis in Kunming city of Yunnan province, China in July 2021.  The complete genome of PyRV1 was amplified by RT-PCR (with the specific primers designed from the contigs and Sanger-sequencing was used to confirm the HTS data ( Figure S1C and Figure S2). The 5′/3′-ends were determined by RACE ( Figure S1D). All the RT-PCR obtained sequences, including the two ends, were assembled to compile the full-length genome sequence of 13,709 bases. The sequence was deposited in the GenBank databases under accession number OL439478. Pair wise comparison of the complete genome sequence with betanucleorhabdoviruses from GenBank showed that PyRV1 had 55.1% nt sequence identity to SYNV, 53.7% to DYVV, and 53.6% to BCaRV (Table 1). Moreover, PyRV1 was clustered within the genus Betanucleorhabdovirus in a clade with SYNV, DYVV and BCaRV in the phylogenetic tree based on the complete genome sequences of the selected rhabdoviruses (Fig. 2).

Symptoms on
Genome organization of PyRV1. The genome of PyRV1 had an organization similar to SYNV. The genome contains 6 ORFs in the order 3′-N-P-P3-M-G-L-5′, which were similar to most nucleorhabdoviruses. The PyRV1 genome included a 201-nt 3′-leader and a 162-nt 5′-trailer. The 3′-leader and the 5′-trailer of PyRV1sharing 23 complete complementary nucleotides that could potentially form a panhandle structure common to all known rhabdovirus genomes. The 3′-leader of PyRV1 had 17 nt and 12 nt identities to those of ZPNRV and SYNV, respectively (Fig. 3). The PyRV1 ORFs were separated by highly conserved intergenic region, which were composed of a polyadenylation signal of the preceding gene, non-transcribed intergenic spacer and transcriptional start of the following gene (    Table 1). The N protein contains a cytoplasmic and nuclear localization signal (NLS) (cNLS) at aa 484-494, and a leucine-rich nuclear export signal (NES) was predicted at positions 39 ( Table 3) (Table 1). In addition, only the G protein contained a predicted transmembrane domain at aa 552-574 (GLFGGIAKVFILIICCIIVYI) and a signal peptide site at aa1-25 (e = 0.7273). The NLS and NES predictions for each ORF aa sequence indicate that PyRV1 may replicate in the nucleus of infected cells.
Conserved residues and motifs. The amino acid sequences of the predicted PyRV1 proteins were aligned with those of available plant rhabdoviruses. The alignment of conserved residues and motifs revealed that PyRV1 shared some conserved residues and motifs in N, G and L proteins with other nuleorhabdoviruses. No conserved residues were identified in P, P3 and M between PyRV1 and other plant rhabdoviruses. Thirteen conserved positions and one motif (364-366,WKY) were identified in the N protein, 10 conserved cysteine residues (79, 83,    Table 3. Analysis of PyRV1 sequence for nuclear localization signals (NLS), nuclear export signals, transmembrane domains and Signal peptite. Phylogenetic analysis of the complete genome and predicted proteins. To understand phylogenetic relationship between PyRV1 and other plant rhabdoviruses, the maximum likelihood phylogenic trees were generated using MEGA X program. The phylogenetic tree based on complete betanucleorhabdoviral genome inferred that PyRV1 is most closely related to SYNV, with at high bootstrap value > 88 (Fig. 2). The phylogenetic tree of the deduced aa sequences of L proteins of the Betarhabdovirinae sub-family also indicated the closest relative of PyRV1 was SYNV. These viruses formed a cluster clearly separated from other betanucleorhabdoviruses, alphanucleorhabdoviruses, gamanucleorhabdovirus, cytorhabdoviruses, dichorhaviruses and varicosaviruses formed separate clusters well separated from each other ( Figure S3) as would be expected. Phylogenetic relationships for all of the other five proteins also showed that PyRV1 clustered together with SYNV, CdVCV, BCaRV, DYVV and SYVV of the genus Betanucleorhabdovirus (not shown).
RT-PCR detection for field samples. Ten field samples, collected from the plantation of P. polyphylla var.
yunnanensis were detected by using PyRV1 specific primer derived from L conversed motifs. A 700-bp specific PCR product from one sample was amplified ( Figure S4). The amplicon was sequenced to verify the infection of PyRV1 to this sample (data not shown).

Discussion
The presence of a novel nucleorhabdovirus in P. polyphylla var. yunnanensis was established by using EM and HTS technologies. The morphological studies by TEM confirmed that the etiologic agent was associated with a rhabdovirus. The morphological results were verified by HTS and targeted amplicon sequencing. The genome of PyRV1 was organized similarly to those of SYNV, SYVV, DYVV, BCaRV and bird's-foot trefoil-associated virus 1 (BFTV-1). The genome contained 6 genes in the order 3′-N-P-P3-M-G-L-5′, each gene being separated by a conserved IGR (UUC UUU UU GG UUG), that was common to SYNV ( Table 2). The genome nucleotide sequence was observed to share approximately 41.3% to 55.1% identities (Table 1) between PyRV1 and other betanucleorhabdoviruses. Sequence identities of all ORFs provided evidence that PyRV1was the most similar to the respective counterparts in SYNV, DVYY, SYVV, BCaRV, ZPNRV and CdVCV of the genus Betanucleorhabdovirus. Phylogenetic analysis based on the complete genome, and aa sequences of the L and N proteins showed PyRV1 clustered within the branch of betanucleorhabdoviruses including SYNV, BCaRV, DYVV, SYVV, CdVCV ( Fig. 2 and S3). Nucleorhabdoviruses (genera Alphanucleorhabdovirus, Betanucleorhabdovirus, Gammanucleorhabdovirus) replicate in viroplasms in the host cell nucleus 13,35 . As has been reported for both SYNV and DYVV 36,37 , the NLS and NES were also observed in the encoded proteins of PyRV1 (Table 3). The PyRV1 P3 protein, a putative movement protein contained a predicted NLS, but no NES [38][39][40] . The presence of conserved residues and motifs in the N, G, and L proteins, especially the canonical GHP motif Pre-motif A, motif A, B, C, and D in the L protein suggested that they have similar respective functions and/or structural features among plant rhabdoviruses 32,41 . Consequently, complete genome alignments among PyRV1 and other available plant rhabdoviruses showed extremely divergent of these nt and aa sequences, as it is commonly observed for different plant rhabdoviruses 13,31 . Based on the molecular aspects, especially the highest nucleotide sequence identity of 55.1% between PyRV1 and other plant rhabdoviruses is lower than the identity threshold level (75%) for establishing a new species of the genus Betanucleorhabdovirus. Therefore, PyRV1 should be considered as a new species in the genus Betanucleorhabdovirus.
In addition, a new species assigned to the genus Betanucleorhabdovirus has other two characteristics: should occupy different ecological niches (differences in hosts and/or arthropod vectors), can be clearly distinguished in serological tests or by nucleic acid hybridization 15 . Attempts were taken to isolate the virus from single local lesion through inoculation into Chenopodium quinoa leaves but could not possible. So, the virus was directly inoculated into N. benthamiana and N. glutinosa. The inoculated N. benthamiana and N. glutinosa plants exhibited vein clearing symptom that was similar to the symptoms on P. polyphylla var. yunnanensis. Inoculation followed by verification of amplicon sequencing. The virus was mechanically smeared with the pestle onto 1-year seedlings of P. polyphylla var. yunnanensis. The inoculated plants wilted after 1 week. It was not possible to detect virus from the withered leaves. When the virus was inoculated into more than 1-year aged P. polyphylla var. yunnanensis plants, did not exhibit the virial symptoms and could not detect the virus. In nature, plant rhabdoviruses are transmitted by insect vectors such aphids, leafhoppers or planthoppers 21 . SYNV is transmitted by aphid. So, PyRV1 may also be transmitted by aphid. However, we could not investigate the aphid in the diseased field. So, we are not sure about the vector. As a wild plant, P. polyphylla var. yunnanensis has been cultivated in a large scale for more than 20 years, several viruses have been detected in P. polyphylla var. yunnanensis [7][8][9][10][11][12] . However, back inoculation into P. polyphylla var. yunnanensis is a dilemma. The leaves of inoculated P. polyphylla var. yunnanensis exhibited clear vein and yellow symptoms similar to naturally infection (Fig. S5), and a weak targeted fragment also was amplified from inoculated leaves of P. polyphylla var. yunnanensis inoculated by PyRV1 (Fig. S6), however, no particle was observed in the saps of the inoculated leaves. Medicinal plant hosts like Paris sp., Panax sp. and Polygonutum sp. plants may pose additional problems in fulfilling the Koch's postulate by smearing inoculation because the abundant secondary metabolites such as saponins and polysaccharides, which may possess the potential antiviral activities present in the leaf mesophyll, interfere with virus infection 3 www.nature.com/scientificreports/ In the present study, a novel betanucleorhabdovirus (PyRV1) causing vein clearing and leaf crinkle disease was discovered in P. polyphylla var. yunnanensis, and was characterized based on morphological and molecular aspects. This virus was transferred to N. bethamiana and N. glutinosa by mechanical smearing inoculation. However, back inoculation to P. polyphylla var. yunnanensis by insect vector is needed to fulfill the Koch's postulate. Thus, further research is needed to identify natural vectors of this virus as well as alternative hosts, develop a serological assay technique and fluorescent viral protein localization to provide strong evidences of species demarcation.

Conclusions
This study identified a novel negative-sense ssRNA virus of paris yunnanensis nucleorhabdovirus (PyRV1), which we suggest belonged to a new species in the genus Betanucleorhabdovirus, based on the study of morphology and analysis of genomic organization, sequence similarity, and phylogeny. Our results also revealed the significant diversities between PyRV1 and other nucleorhabdoviruses in terms of gene sequences. Further study was needed to characterize this virus in terms of host range, morphogenesis and its insect vectors.

Data availability
All the data presented in this study are available in this article and Supplementary Materials.