Complete genome sequence of a previously undescribed monopartite begomovirus and betasatellite infecting Malvastrum coromandelianum in Cambodia

A previously undescribed monopartite begomovirus was identified in Kampot province, Cambodia, in Malvastrum coromandelianum plants exhibiting yellow vein symptoms characteristic of begomovirus infections. The apparently full-length viral component was cloned and sequenced following enrichment of circular DNA by rolling-circle amplification and restriction enzyme digestion. The genome of the virus was 2737 nucleotides in length (KP188831) and exhibited an organization like that of other monopartite begomoviruses, sharing the highest nucleotide sequence similarity (87.7% identity) with ageratum yellow vein virus (AM940137). A satellite molecule was amplified from total DNA by PCR amplification, using the betasatellite-specific primer pair β01/β02. The satellite molecule (1346 nt, KP188832) had structural characteristics like those of other betasatellites associated with begomoviruses and shared the highest nucleotide sequence similarity (84.8% identity) with malvastrum yellow vein betasatellite (MN205547). According to the criteria established for species demarcation for classification of begomoviruses (family Geminiviridae) and betasatellites (family Tolecusatellitidae), respectively, the virus isolate from M. coromandelianum in Cambodia is a previously undescribed novel monopartite begomovirus, for which the name “malvastrum yellow vein Cambodia virus” (MaYVCV) is proposed, and the betasatellite is a previously undescribed novel betasatellite, for which the name “malvastrum yellow vein Cambodia betasatellite” (MaYVKHB) is proposed.

Plant viruses of the family Geminiviridae have a circular, single-stranded DNA genome encapsidated in a twinned icosahedral particle. They are important global pathogens that cause serious yield losses in many crops. The family includes nine genera, namely Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, and Turncurtovirus, based on genome organization, insect vector, and host range [1,2]. The genus Begomovirus (family Geminiviridae) is the largest group within the family, comprising at least 424 species recognized by the International Committee on Taxonomy of Viruses (ICTV) (http://www.ictvo nline .org/virus Taxon omy.asp), and economically, it is the most important, causing yield losses in many crops, including cassava, cotton, and tomato. Begomoviruses are widely distributed in tropical and subtropical regions of the world and are transmitted exclusively by the whitefly Bemisia tabaci (Gennadius). They can have either a monopartite (DNA-A) or bipartite (DNA-A and DNA-B) genome configurations, depending on the number of genome components they possess [3]. The begomoviral DNA-A or DNA-B component is ~2.6-2.8 kb in size, while the monopartite genome is about 2.8 kb in size. The DNA-A and monopartite genomes encode mostly functionally comparable viral proteins: AV1 and AV2 on the virion-sense strand and AC1, AC2, AC3 and AC4 on the complementary strand, for replication, control of gene expression and encapsidation. DNA-B encodes BV1 and BV2 for movement of the virus in plant cells [4]. Many satellite DNA molecules of approximately 1.3 kb in size have been discovered associated with begomoviruses. These satellite DNA molecules are classified as either betasatellites (genus Betasatellite, family Tolecusatellitidae), or alphasatellites (family Alphasatellitidae). Some satellite DNA molecules of approximately 0.7 kb Handling Editor: Jesús Navas-Castillo.

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in size have been discovered associated with begomoviruses and classified as deltasatellites (genus Deltasatellite, family Tolecusatellitidae) [5,6]. Betasatellites are involved in the development of disease symptoms, a characteristic that is attributed to their function as a suppressor of host plant gene silencing. Begomovirus-betasatellite complexes cause important diseases of vegetables, fiber crops, and ornamental plants and infect many uncultivated wild plant species [7].
In October 2014, three leaf samples, Ca-1, Ca-2 and Ca-3, were collected from three different M. coromandelianum plants exhibiting yellow vein symptoms in Kampot Province, Cambodia ( Fig. 1). Total DNA was extracted using an Easy-Pure Plant DNA Kit (TransGen Biotech, Beijing, China). To detect the suspected begomovirus, PCR amplification was carried out using degenerate primers to amplify the coat protein gene (cp) (AV 494 /CoPR) [16,17] with an expected amplicon size of ~570 bp. An amplicon of the expected size was obtained from all three samples. Sequencing of the amplicon from each sample confirmed the presence of a begomovirus in the symptomatic M. coromandelianum plants based on the closest matches of the sequences in a BLASTn search of the GenBank database with ageratum yellow vein virus [AM940137], with 95.61% nt sequence identity and 100 % coverage.
The amplicons were gel-purified and ligated into the plasmid vector pMD19-T (Takara Biomedical Technology Co., Beijing, China). All plasmids were introduced into Escherichia coli DH5α and sequenced (Invitrogen Co., Shanghai, China). The DNA sequences were assembled, edited, and analyzed using DNAStar software version 5.0 (DNASTAR Inc., Madison, WI, USA). Preliminary identification of the begomovirus was done by comparing its sequence to sequences available in the GenBank database using BLASTn (www.ncbi.nlm.nih.gov) [21]. The pairwise nucleotide (nt) sequence identity was determined using a MUSCLE alignment and the Sequence Demarcation Tool (SDT 1.2) [22].
The insert sequences of three clones, Ca-1, Ca-2 and Ca-3 (2,737 nt), from RCA products digested with BamHI   Based on SDT analysis, the pairwise nt sequence identity values ranged from 69.9 to 87.7% in comparisons of the viral component from this study to those of 21 other closely related begomoviruses available in the GenBank database. The viral component shared the highest nt sequence similarity (87.7% identity) with ageratum yellow vein virus (AYVV, AM940137) and Malvastrum leaf curl Philippines virus (MaLCPHV, KC577540) (87.5% identity). Based on the species demarcation threshold for begomoviruses (91% nt sequence identity) [15], this virus isolated from M. coromandelianum in Cambodia is a previously undescribed begomovirus, for which the name "malvastrum yellow vein Cambodia virus" (MaYVCV) is proposed.
Recombination analysis was carried out with default settings in Recombination Detection Program (RDP) 4.0, using the GENECOV, Max Chi, RDP, Bootscan, Chimaera, 3Seq, and SiSan algorithms [24], but no recombination events were detected in MaYVCV.
The betasatellite was 1,346 nt in length (GenBank accession no. KP188832) and had the typical structure of a betasatellite, with a single ORF (βC1) located on the complementary-sense strand, an A-rich region, and a satellite conserved region (SCR) containing a predicted stem-loop structure with the nonanucleotide sequence TAA TAT TAC [7]. Based on SDT analysis, the pairwise nucleotide sequence identity between the betasatellite and betasatellites available in GenBank was 59.1-84.8%. It shared the highest nt sequence similarity (84.8% identity) with malvastrum yellow vein betasatellite from China (MaYVB, MN205547). Phylogenetic analysis indicated that it clustered with MaYVB (MN205547) to form a unique clade (Fig. 2B). Thus, according to the species demarcation threshold for betasatellites (91% nt sequence identity) [25], this is a previously undescribed betasatellite, for which the name "malvastrum yellow vein Cambodia betasatellite" (MaYVKHB) is proposed.
Alphasatellites infecting malvaceous species have been reported throughout Asia, and also in Burkina Faso, Cameroon, Egypt, Kenya, and Mali [26], and deltasatellites infecting M. coromandelianum plants have been reported in Cuba [5]. However, these DNA satellites were not found in the analyzed samples.
Wild plant hosts of viruses are known to be important reservoirs of begomoviruses and are expected to contribute to viral evolution and to the spread of viruses in cultivated crops. The identification and molecular characterization of viral genomes in wild plants and weeds contributes to our understanding of the genetic diversity, ecology, and evolution of begomoviruses. For example, malvastrum leaf curl virus has been identified infecting M. coromandelianum and was also detected in the cultivated fruit crop plant papaya [27], suggesting the wild host may serve as an important reservoir for the virus. Additional research is required to determine the extent of MaYVCV spread between M. coromandelianum and papaya and other cultivated plant species in Cambodia.