Characterization of a novel Tombusviridae species isolated from Paris polyphylla var. yunnanensis

A novel virus, Paris virus 2 (ParV2), was isolated from diseased Paris polyphylla var. yunnanensis, and its complete genome sequence was determined and analyzed. ParV2 is a positive-sense single-stranded RNA (+ssRNA) virus with a genome size of 4,118 nucleotides. The ParV2 genome contains six putative open reading frames (ORFs) that encode proteins with predicted molecular weights of 40.14, 100.26, 7.31, 7.85, 26.09, and 8.77 kDa. The first ORF (ORF1) of ParV2 encodes a putative protein of 40.14 kDa (P40, nt: 20-1,096), whiles the second ORF (ORF2, 888 aa) containing the GDD motif encodes the highly conserved RNA-dependent RNA polymerase protein (RdRP, nt:20-2,683, P100, 100.26 kDa) of viruses in the family Tombusviridae. Multiple sequence alignments analysis showed that the complete genome sequence of ParV2 shares 31.7-55.5% nucleotide sequence identities with viruses in the family Tombusviridae. Ginger chlorotic fleck-associated tombusvirus (GCFaV-1, Accession No. QKE30557) had the highest sequence identity (55.5%) with ParV2. GCFaV-1 also shares 59.2% RdRP and 34.9% CP amino acid sequence identities with ParV2. Sequence comparisons and phylogenetic analysis of RdRP suggested that ParV2 is a novel member of the family Tombusviridae, and its closest known relative is GCFaV-1.


Introduction
Paris polyphylla var. yunnanensis is a perennial herb that belongs to the genus Paris of the family Trilliaceae. It is widely distributed in Yunnan province, China. Due to its wide medicinal applications, it is used as one of the raw materials for various Chinese patent medicines. The scarcity of wild natural resources and large market demand of P. polyphylla var. yunnanensis as a result of its medicinal importance have led to the rapid development of its artificial planting industry -a major cause of the continual emergence of new diseases. Reports indicate that Paris mosaic necrosis virus (PMNV), Paris polyphylla virus X (PPVX), Pepper mild mottle virus (PMMoV), Cowpea aphid borne mosaic virus (CABMV), and Paris virus 1 (ParV1) infect and cause Handling Editor: Robert H.A. Coutts. Lu Chen and Rex Frimpong Anane equally contributed to this study. viral diseases in P. polyphylla var. yunnanensis, leading to significant losses in plant yield [1][2][3][4][5][6].
Viruses in the family Tombusviridae are positive-sense single-stranded RNA viruses that exhibit smooth or granular appearance with a diameter of 28-35 nm. The RNA of Tombusviridae is encapsulated in an icosahedral (T=3) capsid that is composed of 180 identical protein subunits in three conformationally distinct states (A, B, C). With the exception of Dianthoviruses whose genome is bipartite, all members of Tombusviridae have a non-segmented (monopartite) linear genome of about 3.7-4.8 kb in size. The Tombusviridae consist of 16 genera that is divided into three subfamilies. All members of this family are readily transmitted by mechanical inoculation and through plant material used for propagation or grafting, and both the virion and the genetic material alone are infective. Most Tombusviridae species can be transmitted through the soil either dependent on, or independent of, a biological vector. Although, members of Tombusviridae can either infect monocotyledonous or dicotyledonous plants, no species has been found to infect both.
The experimental host range is wide, but the natural host range of individual virus species is relatively narrow. Typical characteristics of viral diseases caused by the viruses of Tombusviridae include mottling, crinkling, necrosis and deformation of leaf, and some infections are symptomless in their natural hosts. Although variability exists in the number and location of genes within members of the family, they all have a conserved organizational feature. This unifying feature is a highly conserved polymerase that contains the canonical "GDD" motif.
We previously reported a novel potyvirus, Paris virus 1 (ParV1), isolated from diseased P. polyphylla var. yunnanensis leaves exhibiting mosaic and mottle symptoms [6]. In the present study, we identified and characterized the complete genome sequence of a novel virus (ParV2) infecting P. polyphylla var. yunnanensis and confirmed its taxonomic and phylogenic relationship with other known viruses.

Materials and methods
Diseased leaf samples exhibiting leaf mosaic and chlorotic symptoms, and suspected of viral infection ( Fig 1A) were sent to Oebiotech Co. Ltd (Shanghai, China) for high throughput sequencing (HTS) on the Illumina HiSeq 2500 platform. Total RNA was extracted from diseased leaf samples using OMEGA ® Plant RNA Kit (TaKaRa Bioengineering, Dalian) by following manufacturer's instructions. To confirm the sequencing results, total RNA isolated from virus-infected leaves was reverse transcribed into cDNA using the ABM ® 5X All-In-One RT MasterMix (Macklin Biochemical Co. Ltd, Shanghai). Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed using 2×Taq PCR MasterMix (Biomed Gene Technology Co. Ltd., Beijing, China) and the specific primers ParV2:1-13 F/R designed based on the sequence of the assembled contigs to confirm the presence of the virus, and also amplify the whole genome sequence. The 5' and 3' terminals of the viral genome were amplified using 5' RACE and 3' RACE kit (Sangon Biotech, Shanghai; order No. B605102 and B605101) and specific reverse transcription primers 2R, R1, R2, F1 and F2. The sequences of all primers used in this study have been listed in Supplementary Tables S1 and S2. To confirm the classification status of the virus and determine its relationship with other viruses, ORFs in the viral genome sequence and protein molecular masses were predicted by NCBI ORF finder (www. ncbi. nlm. nih. gov/ proje cts/ gorf). The nucleotide (nt) and protein amino acid (aa) sequences of related viruses were retrieved from NCBI database and used for sequence alignments. Sequence identities were calculated using the ClustalW algorithm in BioEdit 7.0 program. Phylogenetic relationships between the novel virus and related viruses were determined using the Neighbor-Joining method (NJ) within the MEGA7.0 program with replicas bootstrapped to 1000.
Further pairwise comparison of the complete genome nucleotide sequence and ORFs of ParV2 indicated that ParV2 share 31.7-55.5% complete genome nt, 26.3-59.2% RdRP, 10.5-42.8% MP1, 7.6-47.2% MP2, and 7.7-34.9% CP aa identities with 21 virus isolates of the family Tombusviridae ( Table 1). The highest identity (55.5%) was seen with GCFaV-1 (QKE30557) followed by members of the genus Panicovirus (40.6-43.7%). Again, the highest aa identities of the individual proteins of ParV2 were seen with GCFaV-1, followed by Machlomovirus (RdRP and MP1) or Panicovirus (MP2 and CP). These results indicate that ParV2 is a member of the Tombusviridae, although it may not have all the characteristics of specific genera within the family Tombusviridae.
To determine the phylogenetic status of ParV2 and the evolutionary relationship between ParV2 and other viruses of the family Tombusviridae, phylogenetic tree was constructed using the aa sequences of RdRP protein (a reliable indicator for virus taxonomy). It can be seen from the tree that ParV2 clustered with the unassigned/unclassified GCFaV-1, and machlomoviruses and panicoviruses of the subfamily Procedovirinae. GCFaV-1 is the closest known relative of ParV2 (Fig. 2). It can be inferred from the tree that ParV2 shares a recent common ancestor with viruses of the genera Machlomovirus and Panicovirus, although it could not be placed within any of these genera. It therefore suggest that, ParV2 and GCFaV-1 may have diverged from the branch that gave rise to the genera Machlomovirus and Panicovirus (Fig. 2). It can be concluded that ParV2 is a member of the subfamily Procedovirinae of the family Tombusviridae, although it couldn't be allocated to any Procedovirinae-specific genera. These results confirm our pairwise comparison, and genome analyses results which indicated that ParV2 is a novel member of the family Tombusviridae. Fig. 2 Phylogenetic analysis of ParV2. Phylogenetic tree was constructed using the RdRP amino acid sequence of ParV2 and 58 other species of Tombusviridae. The tree was constructed with MEGA 7.0 using the Neighbor Joining Method with 1000 bootstrap replicates. Values at the branches refer to bootstrap values (%). Individual accession numbers precede each Tombusviridae species name and members of different genera have been indicated in braces. The scale bar at the lower left corresponds to a genetic distance of 0.1.

Discussion
In this study, we identified and characterized a novel plant virus that belongs to the family Tombusviridae, and is tentatively named Paris virus 2. ParV2 has a typical Tombusviridae genome organization [19][20][21][22]. Multiple sequence alignments analysis showed that ParV2 share 31.7-55.5% nt sequence identities with members of the family Tombusviridae. Although, ParV2 clustered with viruses in the genera Machlomovirus and Panicovirus, the closest known relative of ParV2 is the unclassified/unassigned GCFaV-1, which showed the highest sequence similarity with ParV2. Similar to machlomoviruses and panicoviruses genomes that encode a 7-8 kDa MP1 and 6-9 kDa MP2, ParV2 genome also encode an MP1 and MP2 of 7.31 kDa and 7.85 kDa respectively. In contrast, genomes of tombusviruses and aureusviruses encode a conserved 22-27 kDa MP, whiles that of dianthoviruses encode another type of MP of about 34 kDa. These results indicate the relationship of ParV2 with viruses of the genera Machlomovirus and Panicovirus.
One of two distinct groups of CP subunit always exist in the capsids of Tombusviridae species. In one group of genera (Machlomovirus, Necrovirus and Panicovirus) that have structured CP lacking P domain, the CP subunit ranges in size of 25-30 kDa, whiles in the other group of genera with a CP-containing P domain, the CP subunit ranges in size of 37-48 kDa. Our comparative sequence analysis results revealed that CP of ParV2 has a size of 26.09 kDa, that is similar to the structured CP of the genera Machlomovirus, Necrovirus and Panicovirus. The genomic RNA of Machlomovirus and Panicovirus are 4.4 kb (encoding four ORFs) and 4.3 kb (encoding five ORFs) respectively. In contrast, ParV2 and GCFaV-1 have genomic RNA of 4.1kb that encode six ORFs. These data suggest that although ParV2 and GCFaV-1 share various characteristics with Machlomovirus and Panicovirus species, ParV2 and GCFaV-1 belong to a distinct genus. These results also confirm our phylogenetic data that indicated that GCFaV-1 is the closest known relative of ParV2, and that ParV2 and GCFaV-1 share a recent common ancestor with the genera Machlomovirus and Panicovirus.
Taken together, ParV2 should be considered a new species of the family Tombusviridae, and its taxonomic status should be further studied. We therefore propose that ParV2 and GCFaV-1 should be assigned to a new genus within the subfamily Procedovirinae in the family Tombusviridae. In addition to the viral-symptoms, there were obvious insect bites on the infected leaves of P. polyphylla var. yunnanensis. It is possible that the virus infected the plants through insect transmission. This is the first report of a virus of Tombusviridae infecting P. polyphylla var. yunnanensis. As a new virus, further studies are required to ascertain its method of transmission and possible vector.