Identification, Molecular and Phylogenetic Characterization of Two Isolates of vinca mosaic virus Based on p1 and cp Genes

 Vinca minor or lesser periwinkle is a perennial, herbaceous and creeping plant belonging to the genus Vinca and the family Apocynaceae. In addition to being an ornamental and cover plant, V. minor is a valuable herbal plant in traditional medicine, and it also acts as a natural source for the industrial manufacture of brain blood flow stimulants. Periwinkle contains more than 150  alkaloids, which have been isolated from the aerial parts and roots of the plant, so far (Proksa et al.,1988), consisting of vincaminorine, vincaminoreine, minovine, minovincine, vincamine, which has antihypoxic and neuroprotective properties as well as modulatory effects on brain circulation and neuronal homeostasis (Farahanikia et al., 2011). Vinca mosaic virus (VMV), belonging to the genus Potyvirus and the family Potyviridae, causes severe to mild mosaic symptoms, yellowing, blistering and leaf curl in the leaves of the periwinkle plant. In this study, the molecular and phylogenetic characteristics of two isolates of VMV were investigated via analysis of p1 and cp genes.

 Virus-like symptoms, such as mosaic and mottling, yellowing, blistering and leaf curling were found on the leaves of V. minor plants in the pardis campus of Ferdowsi University of Mashhad, Razavi Khorasan, Iran, during a survey in September 2019. Total RNA was extracted from the leaves with the SV Total RNA Isolation Kit to determine the causal agent(s) (Promega, USA). Deep sequencing was carried out on the samples by Macrogen Company in South Korea. Analysis of the results with CLC Genomics Workbench v.12.0.3 software showed that these plants were infected with two isolates of VMV. The cp and p1 gene sequences of VMV isolates were determined. Clustal Omega software was used to compare multiple sequence alignments among sequences and determine the percentage of identities at the nucleotide and amino acid levels. To determine phylogenetic relationships and evolutionary origin of Iranian VMV isolates, phylogenetic tree was drawn based on nucleotide and amino acid sequences of cp and p1 genes using MEGA 7 software and Maximum-Likelihood (ML) method with 1000 replications (Bootstrap). The SDT software (v.1.2) and Muscle sequencing were used to plot the similarity matrix among the isolates at the nucleotide and amino acid levels.

 The identities between the cp gene of two Iranian VMV isolates (VMV-IR-1 and VMV-IR-2) at the nucleotide (nt) and amino acid (aa) levels were 93.11% and 97.00% respectively. For the p1 gene, the obtained results showed 68.35% identity at the nt level and 97.00% at the aa level. The highest percent of nt identity of the VMV cp with other potyviruses in the GenBank was with ASV1 (asparagus virus 1) (75.00% with VMV-IR-1 and 73.82% with VMV-IR-2) VMV-IR-1 and VMV-IR-2 showed the highest aa identity with VDMV (vanilla distortion mosaic virus) (70.39%). The highest nt and aa identity of p1 gene of VMV isolates was with CatMV (catharanthus mosaic virus) (52.29% VMV-IR-1 and 51.31% with VMV-IR-2) and ZTMV (zucchini tigre mosaic virus) (28.70%), respectively. Protected motifs were also identified in the cp and p1 gene sequences. Dendrograms obtained from phylogenetic analysis based on nt and aa acid sequences of these genes placed two Iranian VMV isolates, along with other species of potyviruses in the GenBank, viruses in two and three separate groups respectively. Based on cp gene sequences, at the aa level Iranian VMV isolates is most closely related to yam mosaic virus (YMV) virus, and at the nt level with the closest viruses are include sweet potato virus 2 (SPV2), sugarcane mosaic virus (SCMV), onion yellow dwarf virus (OYDV) and johnsongrass mosaic virus (JGMV) in the same group. Based on the p1 gene sequences, VMV isolates at the nt level were most closely related to the JGMV virus and at the aa level were most closely related to the YMV and celery mosaic virus (CeMV) viruses.

 In this study, for the first time, the molecular properties of VMV isolates were determined based on the cp and p1 genes, and the phylogenetic position of two Iranian VMV isolates was drown. The results showed that the cp gene can be used for taxonomic purposes. Considering the medicinal and ornamental properties of periwinkle and effect of the viruses on its properties, special attention should be paid to viral diseases of this plant.