Complete genome analysis of a novel recombinant isolate of pepper veinal mottle virus from mainland China
© Zhang et al. 2015
Received: 1 September 2015
Accepted: 8 November 2015
Published: 16 November 2015
Pepper veinal mottle virus (PVMV) was well established in Africa, and also reported infecting pepper (Capsicum annuum L) in Taiwan and India. However, there is not available of PVMV in mainland China. Here, the first complete genome sequence of PVMV isolated from pepper in mainland China was reported.
The complete genomic sequence of isolate PVMV-HN isolated from pepper in mainland China is reported in this study. The genome of PVMV-HN is 9793 nucleotides (nt) excluding the poly (A) tail, shares 98-99 % nucleotide sequence identity with those two PVMV isolates from Ghana and Taiwan. Recombinant analysis showed that PVMV-HN probably represents a novel recombinant of PVMV. The phylogenetic relationship of PVMV-HN isolate to other PVMV isolates and other potyviruses based on genome or polyprotein sequence level and CP gene level, was also analyzed in this study.
The current study will help to understand phylogenetic relationship of isolate PVMV-HN.
Five Potyvirus spp. have been reported to infect pepper crops (Capsicus spp.) , of these, Pepper veinal mottle virus (PVMV), has been considered to be a major constraint to pepper production for yield and fruit quality reduction . The virion of PVMV is flexuous filaments, enveloped a single molecule of linear, positive-sense, single-stranded ribonucleic acid (ssRNA), about 9.7-10 Kb in size, which has a poly (A) tract at the 3’ end, and its 5’ end covalently linked to the virus-encoded VPg protein [3, 4]. PVMV could form viral inclusion bodies and “pinwheels” in the cytoplasm of infected cells, which similar with other potyviruses , however, its serologically unrelated to other several pepper potyviruses, such as Tobacco etch virus (TEV) , Potato virus Y (PVY)  and Chili veinal mottle virus (ChiVMV) , and so on are extensively infecting pepper crops in China.
The first complete genome RNA sequence of PVMV was originally isolated from Capsicus frutescens in Ghana , and subsequently the other complete genome RNA sequence isolated from Taiwan have been deposited in GenBank (No. FJ617225). This virus is well documented of infected pepper crops in several countries in Africa [10, 11], and the phylogenetic relationship among different isolates with CP gene level has been extensively studied . In Asia, this virus was reported infecting pepper crops in India and Taiwan . However, as our best knowledge, there is no complete genome RNA sequence of PVMV infecting pepper crops in mainland China so far. In this study, the full genome sequence of a PVMV isolated from pepper (Capsicum annuum) in Hunan province, China was determined and compared to those of various PVMV isolates and other potyviruses.
Viral material and sequence analysis
The viral symptoms of the pepper sample display mottle, yellowing and malformation on leaf, this pepper plant was sampled in a survey of pepper diseases in Hunan province, China in 2014. Total RNA infected pepper sample was extracted by an RNeasy mini kit (QIAGEN, Germany). Full-length cDNA was synthesized by using an oligo(dT)18 primer and TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for PCR Kit (TransGen Biotech, Beijing,China) according to manufacturer’s introduction. The complete genome sequence of the isolate PVMV-HN was amplified from eight overlapping fragments using specific primers (Additional file 1: Table S1), and the 5’- and 3’-terminal ends of the genome was reconfirmed by 5’-RACE and 3’-RACE, respectively, as described previously . PCR products were cloned into pEASY-T5 Vector (TransGen, Beijing, China) and sequenced by Sangon Biotechnology Co., Ltd. (Shanghai, China) using an ABI3730 automated DNA sequencer (Applied Biosystems, USA). Sequences were assembled using DNAMAN version 8 (Lynnon, Quebec, Canada). Sequences analysis and comparison of PVMV-HN to the reference sequences were performed using DNAStar 7.01 package (DNASTAR, Madison, USA). The complete RNA genome or polyprotein sequences and CP sequences of other PVMV isolates and other Potyviruses were downloaded from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/). The information of all used sequences was listed in Additional file 1: Table S2. Multiple nucleotide sequence alignments were performed with CLUST W, possible recombination events were evaluated by Recombination Detection Program (RDP3)  and a phylogenetic tree was reconstructed by the maximum-likelihood method using MEGA5 .
The complete genome of the PVMV-HN isolate comprises 9793 nucleotides (nt), with 194 nt at the 5’ non-translated region (NTR) and 373 nt at the 3’ NTR. Similar as other potyviruses, a large typical open reading frame (ORF) was identified as a polyprotein containing 3074 amino acids (aa) with an AUG start codon and UGA stop codon. PVM-HN shares 99 % nucleotide sequence identity with PVMV isolate of Ghana (DQ645484), and 98 % with PVMV isolate of Taiwan (FJ617225) with genome level, however, only 94 % nucleotide sequence identity with PVMV isolates (GQ918276 and GQ918274) from Mali with polyprotein gene level.
This study provided the first complete genome sequence about PVMV from mainland China and revealed it is probably a novel recombinant.
Nucleotide sequence accession number
The complete genome sequence of the isolate of PVMV-HN is permanently available in the GenBank database under the accession number KR002568.
This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (no. 201303028) and the Agriculture Research System of China (CARS-25-B-05).
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- Moury B, Palloix A, Caranta C, Gognalons P, Souche S, Gebre Selassie K, et al. Serological, molecular, and pathotype diversity of pepper veinal mottle virus and chili veinal mottle virus. Phytopathol. 2005;95:227–32.View ArticleGoogle Scholar
- Fajinmi AA, Odebode CA. Evaluation of maize/pepper intercropping model in the management of pepper veinal mottle virus, genus potyvirus, family potyviridae on cultivated pepper (Capsicum annuum L.) in Nigeria. Arch Phytopathol Plant Protection. 2010;43(15):1524–33.View ArticleGoogle Scholar
- Brunt AA, Kenten RH. Pepper veinal mottle virus. A new member of the potato virus Y group from peppers (Capsicum annuum L. and C. frutescens L.) in Ghana. Ann Appl Biol. 1971;69:235–43.View ArticleGoogle Scholar
- Barnett OW, Adam G, Brunt AA, Dijkstra J, Dougherty WG, Edwardson JR, et al. Potyviridae family. Arch Virol. 1995;10(Suppl):348–58.Google Scholar
- Gibbs A, Ohshima K. Potyviruses and digital revolution. Annu Rev Phytopathol. 2010;48:10.1–10.19.View ArticleGoogle Scholar
- Daia J, Cheng JL, Huang T, Zheng X, Wu YF. A multiplex reverse transcription PCR assay for simultaneous detection of five tobacco viruses in tobacco plants. J Virol Methods. 2012;183(1):57–62.View ArticleGoogle Scholar
- Tian YP, Liu JL, Zhang CL, Liu YY, Wang B, Li XD, et al. Genetic diversity of potato virus Y infecting tobacco crops in China. Phytopathol. 2011;101(3):377–87.View ArticleGoogle Scholar
- Zhao FF, Xi DH, Liu J, Deng XG, Lin HH. First report of chilli veinal mottle virus infecting tomato (Solanum lycopersicum) in China. Plant Dis. 2014;98(11):1589.View ArticleGoogle Scholar
- Ha JH, Hong JS, Kim TS, Ryu KH. Complete genome sequence of an isolate of pepper veinal mottle virus and phylogenetic relationship with other potyvirus. Arch Virol. 2008;153:2315–8.View ArticlePubMedGoogle Scholar
- Alegbejo MD, Abo ME. Ecology, epidemiology, and control of pepper veinal mottle virus (PVMV), genus potyvirus, in West Africa. J Sust Agric. 2002;20:5–16.View ArticleGoogle Scholar
- Gorsane F, Fakhfakh H, Tourneur C, Makni M, Marrakchi M. Some biological and molecular properties of pepper veinal mottle virus isolates occurring in Tunisia. Plant Mol Biol Rep. 1999;17:149–58.View ArticleGoogle Scholar
- Cheng YH, Wang RY, Chen CC, Chang CA, Jan FJ. First report of Pepper veinal mottle virus in tomato and pepper in Taiwan. Plant Dis. 2009;93(1):107.View ArticleGoogle Scholar
- Martin DP. Recombination detection and analysis using RDP3. Methods Mol Biol. 2009;537:185–205.View ArticlePubMedGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Sztuba-Solińska J, Urbanowicz A, Figlerowicz M, Bujarski JJ. RNA-RNA recombination in plant virus replication and evolution. Annu Rev Phytopathol. 2011;49:415–43.View ArticlePubMedGoogle Scholar