A novel East African monopartite begomovirus-betasatellite complex that infects Vernonia amygdalina

The complete genomes of a monopartite begomovirus (genus Begomovirus, family Geminiviridae) and an associated betasatellite found infecting Vernonia amygdalina Delile (family Compositae) in Uganda were cloned and sequenced. Begomoviruses isolated from two samples showed the highest nucleotide sequence identity (73.1% and 73.2%) to an isolate of the monopartite begomovirus tomato leaf curl Vietnam virus, and betasatellites from the same samples exhibited the highest nucleotide sequence identity (67.1% and 68.2%) to vernonia yellow vein Fujian betasatellite. Following the current taxonomic criteria for begomovirus species demarcation, the isolates sequenced here represent a novel begomovirus species. Based on symptoms observed in the field, we propose the name vernonia crinkle virus (VeCrV) for this novel begomovirus and vernonia crinkle betasatellite (VeCrB) for the associated betasatellite. This is the first report of a monopartite begomovirus-betasatellite complex from Uganda.

Abstract The complete genomes of a monopartite begomovirus (genus Begomovirus, family Geminiviridae) and an associated betasatellite found infecting Vernonia amygdalina Delile (family Compositae) in Uganda were cloned and sequenced. Begomoviruses isolated from two samples showed the highest nucleotide sequence identity (73.1% and 73.2%) to an isolate of the monopartite begomovirus tomato leaf curl Vietnam virus, and betasatellites from the same samples exhibited the highest nucleotide sequence identity (67.1% and 68.2%) to vernonia yellow vein Fujian betasatellite. Following the current taxonomic criteria for begomovirus species demarcation, the isolates sequenced here represent a novel begomovirus species. Based on symptoms observed in the field, we propose the name vernonia crinkle virus (VeCrV) for this novel begomovirus and vernonia crinkle betasatellite (VeCrB) for the associated betasatellite. This is the first report of a monopartite begomovirus-betasatellite complex from Uganda.
The genus Begomovirus is the largest of the seven genera in the plant virus family Geminiviridae [3,19]. Begomoviruses are transmitted by the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) to a large variety of cultivated and wild-plant species [16]. Begomoviruses have a circular, single-stranded DNA genome, monopartite or bipartite, encapsidated in twinned icosahedral particles. Bipartite begomoviruses have two genome components, referred to as DNA-A and DNA-B, of similar size (2.5-2.8 kb), while monopartite begomoviruses have only one component, which is similar to DNA-A of bipartite begomoviruses. The DNA-A virion-sense strand encodes coat (CP) and pre-coat (pre-CP) proteins, the latter of which is present only in Old World (OW) begomoviruses. The DNA-A complementarysense strand encodes the replication-associated protein (Rep), a transcriptional activator protein (TrAP), a replication enhancer protein (REn) and C4 protein. DNA-B encodes a nuclear shuttle protein (NSP) on the virion-sense strand and a movement protein (MP) on the complementary-sense strand. There are more than 300 accepted begomovirus species according to the recently updated demarcation criteria for the genus, which consider a DNA-A pairwise identity of 91% as the species threshold [4]. Recombination is a phenomenon that is crucial for speciation and evolution in the family Geminiviridae and contributes to the richness in species of the genus Begomovirus. This stresses the importance of recombination studies when analysing new begomoviruses.
Several types of DNA satellites have been described to be associated with begomoviruses: betasatellites [1], alphasatellites [2] and deltasatellites [11]. Betasatellites are  1). Morphological identification of the plant samples was confirmed molecularly by DNA barcoding using chloroplast rbcL and matK genes [8]. Total DNA was extracted from leaf tissue using a modified CTAB method [17] and used as a template for rollingcircle amplification (RCA) using u29 DNA polymerase (TempliPhi kit, GE Healthcare). Amplified RCA products were digested with a set of restriction enzymes (BamHI, EcoRI, HindIII, NcoI, NheI and SalI), and both samples generated similar restriction patterns, which suggested the presence of a putative monopartite begomovirus (*2.8 kbp) and a putative DNA satellite (*1.3 kbp) in each sample. PCR was carried out using degenerate primers for DNA-B [18] but none of the samples yielded amplification products. Fragments of RCA products digested with EcoRI (*2.8 kbp) and NcoI (*1.3 kbp) were cloned into pBlueScript II SK (?) (Stratagene) and pGEM-T Easy Vector (Promega), respectively. Recombinant plasmid DNAs were introduced into Escherichia coli DH5a by electroporation, and selected clones were sequenced at Macrogen Inc. (Seoul, South Korea). Initial sequence similarity comparison was performed using the BLAST program (http://www.ncbi.nih.gov/). Sequence alignments were performed using MUSCLE [5], pairwise identity scores were calculated using SDT (Sequence demarcation tool) [15], and MEGA 7 was used for phylogenetic analysis [9].
Recombination is commonly detected in begomovirus and betasatellite genomes [6,10,14]. To detect putative recombinant fragments in the novel genomes, a search for potential parental begomoviruses and betasatellites in the GenBank database was conducted using SWeBLAST [7]  with a window size of 200 and a step size of 200. The sequences with the highest SWeBLAST scores were selected for alignment using MUSCLE [5] and subsequent recombination analysis using the RDP4 package with default settings [12]. This analysis showed the presence of recombinant fragments in both VeCrV and the associated VeCrB (Supplementary Table S1). Interestingly, recombination events detected in VeCrV involve genomes from Asia and Africa.
Phylogenetic analysis showed that VeCrV isolates clustered with two OW begomoviruses from Africa, tobacco leaf curl Zimbabwe virus (AF350330) and tobacco leaf curl Comoros virus (AM701760) (Fig. 2A)