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.ictvonline.org/virusTaxonomy.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 . 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 . 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 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 .
The perennial plant species Malvastrum coromandelianum is native to South America but has been introduced and established in tropical and subtropical regions, including Asia. To date, at least 10 begomoviruses have been found first in M. coromandelianum plants [8-15].
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 EasyPure 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) (AV494/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 complete genome of the putative begomovirus was amplified from one sample by rolling-circle amplification (RCA) (TempliPhi kit; GE Healthcare, Buckinghamshire, UK), followed by digestion with BamHI and HindIII endonucleases (Fermentas, GlenBurnie, MD, USA). The digestion of RCA products with the BamHI restriction enzyme yielded a ~2.7-kbp DNA fragment. The fragments (n = 3) were gel-purified and ligated into the plasmid vector pGEM-3Z (Promega Co., Madison, WI, USA), which had been digested with BamHI, introduced into Escherichia coli DH5α by transformation, and sequenced (Invitrogen Co., Shanghai, China). Total DNA was used as template for PCR amplification with primers for DNA-B components, betasatellites, and alphasatellites (PBLlv2040/PCRc1 , β01/β02 , and DNA101/DNA102 , respectively).
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) . The pairwise nucleotide (nt) sequence identity was determined using a MUSCLE alignment and the Sequence Demarcation Tool (SDT 1.2) .
The insert sequences of three clones, Ca-1, Ca-2 and Ca-3 (2,737 nt), from RCA products digested with BamHI were 99.6%-100% identical to one another. The sequence of Ca-1 was chosen as the representative genome sequence of this begomovirus and deposited in the GenBank database under the accession number KP188831. The sequence had the typical genome organization of a monopartite Old World begomovirus, containing six predicted open reading frames (ORFs).
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) , 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.
Phylogenetic analysis carried out using MEGA  (maximum likelihood with bootstrap values >50% and 1000 iterations) between MaYVCV and 21 other begomoviral DNA-A sequences showed that it clustered with AYVV (AM940137), stachytarpheta leaf curl virus (StaLCuV, AJ564743), MaLCPHV (KC577540), and tomato leaf curl Mindanao virus (ToLCMiV, EU487046) to form a unique clade (Fig. 2A).
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 , but no recombination events were detected in MaYVCV.
The M. coromandelianum samples were tested by PCR for the presence of DNA-B, betasatellites, and alphasatellites, using the primer pairs PBLlv2040/PCRc1 , β01/β02 , and DNA101/DNA102 , respectively. An amplicon of ~1.4 kb was obtained for samples from three diseased plants using β01/β02, but no amplicon was obtained with PBLlv2040/PCRc1 or DNA101/DNA102, suggesting the presence of an associated betasatellite, but no alphasatellite or DNA-B component.
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 TAATATTAC . 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) , 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 , and deltasatellites infecting M. coromandelianum plants have been reported in Cuba . 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 , 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.