Complete genome sequence of an isolate of Pepper veinal mottle virus and phylogenetic relationship with other potyviruses

Abstract

Pepper veinal mottle virus (PVMV) is a virus species in the genus Potyvirus in the family Potyviridae [4]. The virions of members of this genus are flexuous filaments containing a single molecule of linear, positive-sense, single-stranded ribonucleic acid (ssRNA), about 9.7 kb in size, which has a poly (A) tract at the 30-end [1, 3, 12, 18, 20]. The potyviruses are transmitted by aphids in a non-persistent manner and are transmissible experimentally by mechanical inoculation [17]. PVMV, one of pepper-infecting potyviruses, is widespread in African countries and causes veinal yellowing and mottling symptoms in peppers [2, 9] and various weeds such as Physalis angulata [6]. PVMV was first isolated from Capsicum annuum and Capsicum frutescens from Ghana in 1971 [4], and this virus was serologically distinguished as a potyvirus distinct from potato virus Y (PVY) [4]. PVMV causes mosaic and stunt disease in C. frutescens and induces chlorotic local lesions in Chenopodium amaranticolar, Nicotiana tabacum cv. Xanthi and N. tabacum cv. Samsun [2, 4]. The symptoms of infection differ depending on the isolates and host plants [19]. Pepper-infecting potyviruses have been reported from many countries. To date, the properties and complete genome sequences of isolates of Pepper mottle virus (PepMoV), Pepper severe mosaic virus (PepSMV), Chilli veinal mottle virus (ChiVMV), PVMV and various solanaceae-infecting potyviruses (PVY, PTMV, TEV) have been reported [1, 3, 18]. Partial genome sequences of PVMV obtained from various isolates have been reported in some regions [2, 7, 8, 11], but there is no complete genome RNA sequence of PVMV so far. In this study, the full genome sequence and genome structure of a PVMV isolate were determined and compared to those of various PVMV isolates and other potyviruses. A stock culture of PVMV was obtained from the Plant Virus GenBank (Seoul, Korea) which had previously been obtained from the DSMZ (PV-0257, Germany). The isolate was originally isolated from virus-infected C. frutescens in Ghana [4]. The virus used in this study, termed PVMV-P (pepper isolate), was propagated on N. benthamiana. PVMV particles examined by electron microscopy were filamentous, about 770-nm long and 12-nm wide (data not shown), which is the typical morphology described [2]. The virus was purified from virus-infected N. benthamiana leaves by differential centrifugation [7, 12]. Viral genomic RNA was isolated using sodium dodecyl sulphate (SDS)/ proteinase K and phenol extraction method [12, 13] and was used as template for RT-PCR. The RT-PCR was performed in a reaction mixture containing 10 pM of potyvirus-specific primer targeted to the NIb and CP regions, and oligo dT primer [15, 17]. PCR was performed in a thermal cycler for 3 min at 94 C, for a total of 35 cycles (94 C for 40 s, 52 C for 1 min, 72 C for 1 min), and final extension at 72 C for 10 min. The partial sequence of PVMV was determined using the amplified PCR products (GenBank: AB126177). Nucleotide sequences at the 50-end for PVMV were determined by 50-rapid amplification of cDNA ends (RACE) using PVMV 50 RACE (50-GTGAGTGTTGTA GAAGCACGGG-30) and the SMARTII oligo nucleotides (SMART RACE cDNA amplification kit, Clontech, CA, USA). Selected clones and cDNAs were then sequenced J. H. Ha J. S. Hong K. H. Ryu (&) Plant Virus GenBank, Division of Environmental and Life Sciences, Seoul Women’s University, Seoul 139-774, South Korea e-mail: ryu@swu.ac.kr