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

Abstract

Paris yunnanensis, also named as Rhizoma Paridis in the Chinese Pharmacopeia, is a perennial Chinese medicinal herb commonly grown in Southwest China. However, several viruses have been found infecting this plant in recent years. Using high-throughput sequencing (HTS) and Sanger sequencing, this study obtained the complete genome sequences of three capillovirus isolates and one potyvirus isolate. Genomic and phylogenetic analyses revealed that these three capillovirus isolates are the same virus as the newly reported capillovirus, Paris polyphylla chlorotic mottle virus. The newly found potyvirus isolate shares 52.4-68.9% nucleotide sequence identity with other known potyviruses and thus, is grouped into the bean common mosaic virus subgroup. Based on the nucleotide sequence identity, we consider this virus a novel potyvirus species and propose 'Paris potyvirus 5' (ParPV-5) as its common name, and 'Potyvirus shilinense' as its species name. To characterize their biological features, two infectious clones, representing the two viruses, have been constructed through homologous recombination or yeast homologous recombination, and inoculated to several species plants, respectively. The results showed both of the viruses can infect P. yunnanensis and Nicotiana benthamiana. In addition, Paris polyphylla chlorotic mottle virus (PpCMV) can infect N. tabacum var. Xanthi nc, Cucurbita moschata, and Capsicum annuum, and ParPV-5 can infect Cucumis sativus L. and Bidens pilosa L. However, except mild leaf deformation exhibited on the PpCMV-inoculated C. moschata plants, no obvious symptom were observed in these plants including P. yunnanensis. A total of 179 field P. yunnanensis leaf samples from four counties in 2020-2021, and all 640 P. yunnanensis plants from a whole study plot of Lijiang in 2024, were tested using RT-PCR and specific primers, the results showed that PpCMV is a potential preponderant species in some regions, and ParPV-5 has the possible transmission from the original site to other regions.

Keywords: Paris yunnanensis; Biological characters; Infectious cDNA clone; Paris polyphylla chlorotic mottle virus; Paris potyvirus 5.

Fig. 1

Virus-like symptoms on the leaves of P. yunnanensis plants grown in fields: (A) yellowing and necrosis, (B) mottle, (C) leaf edge yellowing, (D) yellowing and blistering.

Fig. 2

Genome organization of Paris polyphylla chlorotic mottle virus (PpCMV) and a schematic showing the infectious clone of PpCMV YLuD isolate. (a) Genome organization of PpCMV. The predicted open reading frames (ORFs) are shown in different colored boxes. The 5’-end and 3’-end untranslated regions (UTR) are represented using solid lines. The conserved domains in the PpCMV polyprotein are viral methyl transferase (Met), viral helicase (Hel), RNA-dependent RNA polymerase (RdRp), and coat protein (CP). (b) A schematic representation showing the strategy used to construct the infectious clone through homologous recombination. Fragment A and B cover the complete genome of PpCMV, amplified through RT-PCR, and inserted into the shuttle vector pCB301 through homologous recombination. (c) Detection of plasmids with correct inserts through PCR. Original gels is presented in Supplementary Figure S4.

Fig. 3

A maximum-likelihood phylogenetic tree constructed using the deduced polyprotein sequences of PpCMV and other representative viruses in the family Betaflxiviridae. The bootstrap analysis was performed using 1000 bootstrap replicates. The scale bar represents a genetic distance of 0.2. Botrytis virus F, a member in the genus Mycolexivirus, family Gammaflexiviridae, was used as an outgroup control. Solid triangles indicate the newly found PpCMV isolates.

Fig. 4

PpCMV-YLuD infectivity and host range assays. (A) The mock- or PpCMV-YLuD-inoculated P. yunnanensis (a), N. benthamiana (b), N. tabacum var. Xanthi nc (c), C. annuum (d) and C. moschata (d) plants. (B) RT-PCR detection PpCMV-YLuD RNA in some inoculated leaves of P. yunnanensis (a), N. benthamiana (b), N. tabacum var. Xanthi nc (c), C. annuum (d), and C. moschata (e). Original gels are presented in Supplementary Figure S5-S9. (C) A TEM micrograph showing PpCMV particles in a systemically infected Paris yunnanensis leaf sample. Bar, 500 nm.

Fig. 5

Genome organization of Paris potyvirus virus-5 (ParPV-5) and a schematic showing construction of ParPV-5 infectious clone. (A) Genome organization of ParPV-5. The 5’- and 3’-end UTRs are represented using solid lines. The ORF is shown using an open box with different colors (matured proteins). The putative PIPO protein is inside the P3 protein and is indicated with a small red box. (B) A schematic showing the yeast homologues recombination (HR)-based clone strategy. Three overlapping ParPV-5 PCR fragments were inserted into the pCB301-2µ-HDV shuttle vector through HR. (C) The mock- or ParPV-5-inoculated B. pilosa L. (a), N. benthamiana (b), P. yunnanensis (c), C. sativus L., RT-PCR detection (e) ParPV-5 in the inoculated leaves of B. pilosa L. (i), N. benthamiana (ii), P. yunnanensis (iii), and C. sativus L. (iiii), Original gels are presented in Supplementary Figure S10-S13. And a TEM micrograph showing viral particles in the systemic leaves of the ParPV-5-inoculated N. benthamiana plants (f). Bar, 200 nm.

Fig. 6

A maximum-likelihood phylogenetic tree was constructed using the deduced polyprotein sequence of ParPV-5 and that of known potyviruses. Bootstrap analysis was applied using 1000 bootstrap replicates. The scale bar represents a genetic distance of 0.2. Sugarcane streak mosaic virus was used as an outgroup control.