Identi cation and characterization of a novel potyvirus infecting Paris yunnanensis

Pingxiu Lan Yunnan Agriculture University: Yunnan Agricultural University Peng He Yunnan Agriculture University: Yunnan Agricultural University Mengji Cao Southwest University Guohua Zhou Chinese Association of Chinese Medicine Li Chenrong Yunnan Agricultural University Guanlin Tan Yunnan Agricultural University Xiaojiao Chen Yunnan Agricultural University Jie Yang Yunnan Agricultural University Taiyun Wei Fujian Agriculture and Forestry University Fan Li (  fanlikm@126.com ) Yunnan Agricultural University https://orcid.org/0000-0002-4394-2431


Introduction
Paris yunnanensis (Chonglou in Chinese), previously recognized as a conspeci c variety of P. polyphylla (P. polyphylla var. yunnanensis), is a perennial herb in genus Paris of family Melanthiaceae in order Liliales [1]. P. yunnanensis is an important Chinese traditional herb and major component material for at least 49 Chinese patent medicines, such as Yunnan Baiyao Powder and Snake-bite Therapeutics. The plant of P. yunnanensis was naturally growing in the 1400-3200 altitude of shady place in Southwest China, but it had been excessively excavated to the edge of extinction and commercially planted to meeting the market demand since 1980s. In 2017, the annual planting area of P. yunnanensis in China was beyond 10000 hm 2 with a production of 50 thousand tons, and the pro t of selling the P. polyphylla related drugs and products got approximately 10 billion CNY (ca. 1.6 billion USD) [1]. However, virus disease became daily serious with its extension of planting years and expansion of planting area. several viral pathogens, including Paris polyphylla virus X (PPVX) [2], Paris mosaic necrosis virus (PMNV) [3], pepper mild mottle virus (PMMoV) [4], Paris virus 1 (ParV1) [5], Paris virus 2 (ParV2) [6] and chilli veinal mottle virus [7] had been reported infecting the cultivation plants of P. yunnanensis and caused production declining. This paper investigated the viral disease of P. yunnanensis in Yunnan province, determined and characterized the complete genome sequence of a novel potyvirus from plants with leaf mottle.

Materials And Method
In August 2017, symptoms of foliar mosaic, mottle and yellowing were observed in a P. yunnanensis commercial plantation of Mangshi, Dehong autonomous prefecture of Yunnan Province (Fig. 1A). Nine leaves samples were collected and pooled into one sample for total RNA extraction and high-throughput sequencing (HTS) by Vazyme Biotech Co., Ltd (Nanjing, China). The quali ed total RNA was depleted ribosomal RNA and then subjected to HTS RAN-seq on the Illumina HiSeq X-ten platform with PE150 bp.
The sequence data were analyzed by CLC Genomic Workbench 9.5 (QIAGEN). A total of 64,278,370 paired-end reads were obtained after removing the failed reads, with which 105,208 contigs larger than 200 bp were assembly generated by de novo. BLASTx analysis of the assembled contigs against the NCBI databases indicated that one large contigs of 9504 nt in length with several uncertain base temporarily marked as "N", was represented the preliminary genome scaffold of a potyvirus and shared the highest amino acid (aa) sequence identity of 51%-52% with Kalanchoe mosaic virus (APX54983).
To con rm the presence of the virus and amplify its whole genome sequence, morphology of the novel potyvirus was observed under transmission electron microscopy (TEM) by negative stain technology.
Speci c primers covering the entire genome sequence were designed according to the 9504 nt contig. All primers have an overlapping region of ~ 115 to 151 nt at the ending of the contiguous amplicons (Table   S1). Total RNAs from the initial 9 samples (0.1 g each) were extracted using EasyPure Plant RNA kit (TransGen Biotech, Beijing, China). RT-PCR was performed using a PrimeScript ™ One-step RT-PCR Kit ver.
2 (TaKaRa Biotechnology Co., Ltd., Dalian, China). The 5'-end sequence was obtained using Multiple alignment was performed using the Clustal Omega Mutiple Sequence Alignment at https://www.ebi.ac.uk/Tools/msa/clustalo/. Nine highly conserved proteolytic cleavage sites in its polyprotein were predicted using a multiple alignment of relevant potyviruses polyprotein according to the criteria proposed by Adams et al [9]. Phylogenetic trees were constructed using maximum likelihood (ML) method in the MEGA 7 software [10].

Results And Analysis
Flexuous, lamentous particles of 700 ~ 800 nm in length were observed by Electron microscopy using crude sap from the virus infected plants (Fig. 1B). The complete genomic sequence of the virus from mangshi (designed as YMSh-CL isolate) was determined to be 9600 nucleotides (nt) excluding the 3'-  (table  1). Phylogenetic analyses were conducted using the deduced polyprotein sequence and selected members of the genus Potyvirus. The virus was clustered as a single clade between the subgroup of turnip mosaic virus (TuMV) and that of PPV ( Fig. 2A). A distinct phylogenetic relationship was maintained when the complete nt was used ( Supplementary Fig. 1), suggesting that the virus should be a divergent species in the genus Potyvirus.  Tables   Table 1 is not available with  protein by a small box in red. The putative proteolytic cleavage sites in the polyprotein and the length in amino acids of each protein is indicated below the genome, whereas the numbers above the genome indicate the start for each region.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Summplmenttable.docx