High-throughput sequencing reveals the presence of novel and known viruses in diseased Paris yunnanensis

Abstract

Paris spp. are important medicinal plant and main raw material for many Chinese patent medicines, but viral diseases have became serious problems in cultivation of this group of important medicinal plants in China. In this study, eight viruses were identified in the diseased plants of Paris yunnanensis by high-throughput sequencing (HTS) and RT–PCR. These viruses include three novel viruses (two potyviruses and one nepovirus), Hippeastrum chlorotic ringspot virus (HCRV), Lychnis mottle virus (LycMoV), Paris mosaic necrosis virus (PMNV), Paris virus 1 and pepper mild mottle virus. The three new viruses were tentatively named Paris potyvirus 3 (ParPV-3), Paris potyvirus 4 (ParPV-4), Paris nepovirus 1 (ParNV-1) and their complete genome sequences were determined. Sequence analyses showed ParPV-3 and ParPV-4 shared the highest amino acid (aa) sequence identities of 54.3% to each other and 53.0–57.8% to other known potyviruses. ParNV-1 had aa sequence identities of 28.8–63.7% at protease-polymerase (Pro-Pol) with other nepoviruses. Phylogenetic analyses further support that the three viruses are new members of their corresponding genera. Analyses of the partial sequences of HCRV and LycMoV infecting P. yunnanensis revealed they diverged from existing isolates by aa sequence identities of 97.1% at glycoprotein precursor of HCRV and 93.3% at polyprotein of LycMoV. These two viruses are reported for the first time in Paris spp. A total of 123 field samples collected from P. yunnanensis in four counties of Yunnan, Southwest China were tested by RT–PCR for detecting each of the eight viruses. Results showed that nearly half of the samples were positive for at least one of the eight viruses. Two potyviruses, ParPV-3 (26.8%) and PMNV (24.4%), were predominant and widely distributed in the fields, while other viruses occurred in low rates and/or had limited distribution. This study insights into the virome infecting P. yunnanensis and provides valuable information for diagnosis and control of viral diseases in P. yunnanensis.

Molecular and biological characterization of infectious full- length cDNA clones of two viruses in Paris yunnanensis, including a novel potyvirus

“…RNA sequencing was performed on an Illumina HiSeq X-ten platform to obtain paired-end reads of 150 bp in length. The obtained raw reads were trimmed, screened for quality, and analyzed using a previously published protocol for plant virus RNA sequencing 7 .The resulting high quality reads were assembled de novo using the CLC Genomics Workbench 20.1 (Qiagen, Germantown, MD, USA). Candidate viral sequence contigs were further analyzed through homology search against the NCBI database using BLASTn and then BLASTx.…”

“…2 (TaKaRa Biotechnology Co., Ltd., Dalian, China) according the manufacturer's protocol. The 5'-end sequences of the two viruses were obtained using the SMARTer®RACE 5'/3' kit (TaKaRa Biotechnology Co. Ltd., Dalian, China), while the 3'-terminus sequences were ampli ed using virus-speci c forward primers and degenerated primers (viral8 and viral9) designed for viruses with poly (A) tails 7 . The resulting PCR products were gel puri ed and cloned into the pMD19-T vector [TaKaRa Biotechnology (Dalian) Co. Ltd., Dalian, China].…”

“…This dramatic change of cultivation method, however, has caused emergences of many virus diseases in P. yunnanensis, leading to a signi cant decrease of rhizome yield and quality. To date, at least 16 viruses in 10 virus genera of nine families have been found in P. yunnanensis (Supplementary Table 1) [7][8][9][10][11][12][13][14][15][16][17][18] . For example, the complete nucleotide (nt) sequence of Paris polyphylla chlorotic mottle virus (PpCMV) was reported during the preparing of this manuscript 18 .…”

“…Cultivation of P. yunnanensis is now a common way to meet the market demand for its rhizome and the maintenance of the wild population. Since 1980s, the cultivation scale of P. yunnanensis has reached to about 10,000 hm 2 in China and the total rhizome yield of 50,000 tons, equivalent to about 1.6 billion USD 7 . This dramatic change of cultivation method, however, has caused emergences of many virus diseases in P. yunnanensis, leading to a signi cant decrease of rhizome yield and quality.…”

Discovery of a new polerovirus infecting Bidens pilosa

“…Four pairs of the virus-speci c primers were also designed to amplify the overlapping viral fragments covering most of the genome of the new virus in RT-PCR. The 5'-terminal sequences was ampli ed using a SMARTer®RACE5'/3' Kit (TaKaRa Biotech Co., Ltd, Dalian, China) while the 3'-terminus was obtained using a virus-speci c forward primer and two degenerated primers of viral8 and Vial9 after adding a poly(A) tail [7]. The PCR products were cloned into the vector pMD19-T (TaKaRa Biotech), and at least three clones were sequenced on both directions for each amplicon (Sangong Biotech Co., Ltd, Shanghai, China).…”