Complete genome sequence analysis of a new potyvirus isolated from Paris polyphylla var. yunnanensis

The complete genome sequence of a new potyvirus from Paris polyphylla var. yunnanensis was determined. Its genomic RNA consists of 9571 nucleotides (nt), excluding the 3’-terminal poly(A) tail, containing the typical open reading frame (ORF) of potyviruses and encoding a putative large polyprotein of 3061 amino acids. The virus shares 54.20%-59.60% nt sequence identity and 51.80%-57.90% amino acid sequence identity with other potyviruses. Proteolytic cleavage sites and conserved motifs of potyviruses were identified in the polyprotein and within individual proteins. Phylogenetic analysis indicated that the virus was most closely related to lily yellow mosaic virus. The results suggest that the virus should be classified as a member of a novel species within the genus Potyvirus, and we have tentatively named this virus “Paris yunnanensis mosaic chlorotic virus” (PyMCV).

Paris polyphylla var. yunnanensis is a perennial herb of the family Liliaceae. It is mainly distributed in southwest China, especially in Yunnan province [1]. Its rhizome contains abundant active components, including steroidal saponins and flavonoids, and has anticancer and haemostatic pharmacological effects. Due to the considerable economic and medicinal value as well as the scarcity of wild resources of P. polyphylla var. yunnanensis, it has been domesticated and widely cultivated in Yunnan. However, cultivated P. polyphylla var. yunnanensis is frequently affected by fungal and viral diseases that lead to a serious loss of yield and a decrease in medicinal value. So far, five viruses have been reported in P. polyphylla var. yunnanensis, including Paris mosaic necrosis virus (PMNV) [2], Paris polyphylla virus X (PpVX) [3], pepper mild mottle virus (PMMoV) [4], chilli veinal mottle virus (ChiVMV) [5], and Paris virus 1 (ParV1) [6], which cause chlorosis, leaf deformation, or yellowing of leaf vein symptoms.
In July 2021, P. polyphylla var. yunnanensis plants exhibiting mosaic and chlorotic symptoms were observed on a paris plantation in Lijiang of Yunnan province, China ( Fig. 1). Leaf samples were collected from the diseased plants, and total RNA was isolated using TRIzol Reagent (Sangon, Shanghai, China). The presence of a potyvirus was confirmed by RT-PCR, using degenerate primers for potyviruses [7,8].
The symptomatic leaves from a single diseased plant were subjected to high-throughput sequencing at TsingKe (Kunming, China). Total RNA from symptomatic leaves were used to construct an rRNA-depleted library using Nextera XT reagents (Illumina, San Diego, CA). The library was sequenced by RNA-Seq, using a NovaSeq 6000 PE150 platform (Illumina, San Diego, CA). A total of 33,126,293 clean reads were obtained using BBmap (https:// github. com/ BioIn foToo ls/ BBMap BBMap -Bushnell B. -sourceforge. net/projects/bbmap/). Of the 83,961 reads composed of plant virus sequences, 77,944 reads (92.83%) were mapped to potyviruses, and the rest showed similarity to sequences from Dahlia mosaic virus. A total of 603,294 contigs were assembled de novo using SPAdes v3.14.1. BLASTx analysis revealed that 27 of the contigs (≥108 bp) mapped to potyviruses. A long contig consisting of 9563 nt shared 57.90% amino acid sequence identity (E-value 0.0, 95%   Fig. S1). Each putative mature protein was compared with other potyvirus proteins in the NCBI database (Supplementary Table S1). The highly conserved motif 2901 GAA AAA AA 2908 and a small ORF (PIPO) encoding 54 aa were detected in the P3 protein of PyMCV ( Supplementary Fig. S2) [9]. Highly conserved potyviral motifs were identified in the PyMCV polyprotein by comparative analysis, including a 608 PTK 610 motif in HC-Pro; 1243 GAVGSGKST 1251 , 1461 VATNIIENGVTL 1472 , 1505 GERIQRLGRVGR 1516 , and 1332 DECH 1335 motifs in CI; and 2590 GNNSGQPSTVVDNT 2603 and 2631 GDD 2633 motifs in NIb. In addition, we discovered an 2805 NAG 2807 motif in the CP that is also found in aphid-transmitted potyviruses [10]. The NAG motif is also present in the CPs of bean yellow mosaic virus (NP734182.1), Mediterranean ruda virus (YP009667146.1), and lily mottle virus (CAD92110.1).
To determine the phylogenetic status of PyMCV and its evolutionary relationship to other viruses of the genus Potyvirus, pairwise comparisons and construction of a phylogenetic tree were performed using a ClustalW multiple alignment in MEGA X [11]. Pairwise comparison analysis showed that the PyMCV polyprotein shared 54.20%-59.60% nucleotide (Supplementary Table S2) and 51.80%-57.90% amino acid sequence (Supplementary Table S3) identity with viruses of the genus Potyvirus. According to the species demarcation criteria for the genus Potyvirus established by the ICTV, members of different species have complete ORF sequences that are generally <76% identical in nucleotide sequence and <82% identical in amino acid sequence [12]. Therefore, PyMCV should be considered a member of a new species in the genus Potyvirus. The maximum-likelihood phylogenetic tree based on polyprotein sequences indicated that lily yellow mottle virus (LYMV) is the closest known relative of PyMCV (Fig. 2).
To protect Paris polyphylla var. yunnanensis from virus infection, further studies are needed to investigate the biological properties of this virus.

Data Availability
The dataset generated during the current study is available in the GenBank database (accession no.ON871824).

Declarations
Conflict of interest All authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.