High molecular diversity and divergent subpopulations of the begomovirus cnidoscolus mosaic leaf deformation virus associated with Cnidoscolus urens.

Begomoviruses have circular, single-stranded DNA genomes encapsidated into twinned quasi-icosahedral particles and are transmitted by whiteflies of the Bemisia tabaci sibling group. Begomoviruses infect cultivated and non-cultivated plants, causing great losses in economically important crops worldwide. To better understand the genetic diversity of begomoviruses infecting the non-cultivated host Cnidoscolus urens, leaf samples exhibiting virus-like symptoms were collected in different localities in the state of Alagoas, Brazil, during 2015 and 2016. Forty-two complete DNA-A sequences were cloned and sequenced by the Sanger method. Based on nucleotide sequence comparisons, the 42 new isolates were identified as the bipartite begomovirus cnidoscolus mosaic leaf deformation virus (CnMLDV). The CnMLDV isolates were clustered in two phylogenetic groups (clusters I and II) corresponding to their sampling areas, and the high value of Wright's F fixation index observed for the DNA-A sequences suggests population structuring. At least seven independent intraspecies recombination events were predicted among CnMLDV isolates, with recombination breakpoints located in the common region (CR) and in the CP and Rep genes. Also, a high per site nucleotide diversity (π) was observed for CnMLDV isolates, with CP being significantly more variable than Rep. Despite the high genetic variability, strong negative or purifying selection was identified as the main selective force acting upon CP and Rep.

Evolutionary dynamics of bipartite begomoviruses revealed by complete genome analysis.

Several key evolutionary events marked the evolution of geminiviruses, culminating with the emergence of divided (bipartite) genomes represented by viruses classified in the genus Begomovirus. This genus represents the most abundant group of multipartite viruses, contributing significantly to the observed abundance of multipartite species in the virosphere. Although aspects related to virus-host interactions and evolutionary dynamics have been extensively studied, the bipartite nature of these viruses has been little explored in evolutionary studies. Here, we performed a parallel evolutionary analysis of the DNA-A and DNA-B segments of New World begomoviruses. A total of 239 full-length DNA-B sequences obtained in this study, combined with 292 DNA-A and 76 DNA-B sequences retrieved from GenBank, were analysed. The results indicate that the DNA-A and DNA-B respond differentially to evolutionary processes, with the DNA-B being more permissive to variation and more prone to recombination than the DNA-A. Although a clear geographic segregation was observed for both segments, differences in the genetic structure between DNA-A and DNA-B were also observed, with cognate segments belonging to distinct genetic clusters. DNA-B coding regions evolve under the same selection pressures than DNA-A coding regions. Together, our results indicate an interplay between reassortment and recombination acting at different levels across distinct subpopulations and segments.

Identification and Characterization of Two Novel Geminiviruses Associated with Paper Mulberry (Broussonetia papyrifera) Leaf Curl Disease.

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

Complete genome sequence of a new bipartite begomovirus infecting Macroptilium lathyroides in Brazil.

A distinct bipartite begomovirus was isolated in northeastern Brazil infecting Macroptilium lathyroides showing symptoms of yellow mosaic. The complete genome (DNA-A and DNA-B) of the virus was cloned using rolling circle amplification and subsequently sequenced. Clones presented the typical genomic organization of a New World bipartite begomovirus. Based on the current taxonomic criteria established for the genus Begomovirus, the virus corresponds to a new species, showing highest nucleotide identity with other Brazilian begomoviruses that infect leguminous hosts. In phylogenetic analysis the virus clustered with bean golden mosaic virus. Recombination events were not detected. We propose the name Macroptilium common mosaic virus (MacCMV) for the virus reported in this study.

Interaction between the New World begomovirus Euphorbia yellow mosaic virus and its associated alphasatellite: effects on infection and transmission by the whitefly Bemisia tabaci.

The majority of Old World monopartite begomoviruses (family Geminiviridae) are associated with satellite DNAs. Alphasatellites are capable of autonomous replication, but depend on the helper virus for movement, encapsidation and transmission by the insect vector. Recently, Euphorbia yellow mosaic alphasatellite (EuYMA) was found in association with Euphorbia yellow mosaic virus (EuYMV) infecting Euphorbia heterophylla plants in Brazil. The geographical range of EuYMA was assessed in a representative sampling of E. heterophylla plants collected in several states of Brazil from 2009 to 2014. Infectious clones were generated and used to assess the phenotype of viral infection in the presence or absence of the alphasatellite in tomato, E. heterophylla, Nicotiana benthamiana, Arabidopsis thaliana and Crotalaria juncea. Phenotypic differences of EuYMV infection in the presence or absence of EuYMA were observed in A. thaliana, N. benthamiana and E. heterophylla. Symptoms were more severe when EuYMV was inoculated in combination with EuYMA in N. benthamiana and E. heterophylla, and the presence of the alphasatellite was determinant for symptom development in A. thaliana. Quantification of EuYMV and EuYMA indicated that EuYMA affects the accumulation of EuYMV during infection on a host-dependent basis. Transmission assays indicated that EuYMA negatively affects the transmission of EuYMV by Bemisia tabaci MEAM1. Together, these results indicate that EuYMA is capable of modulating symptoms, viral accumulation and whitefly transmission of EuYMV, potentially interfering with virus dissemination in the field.

Capulavirus and Grablovirus: two new genera in the family Geminiviridae.

Geminiviruses are plant-infecting single-stranded DNA viruses that occur in most parts of the world. Currently, there are seven genera within the family Geminiviridae (Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus). The rate of discovery of new geminiviruses has increased significantly over the last decade as a result of new molecular tools and approaches (rolling-circle amplification and deep sequencing) that allow for high-throughput workflows. Here, we report the establishment of two new genera: Capulavirus, with four new species (Alfalfa leaf curl virus, Euphorbia caput-medusae latent virus, French bean severe leaf curl virus and Plantago lanceolata latent virus), and Grablovirus, with one new species (Grapevine red blotch virus). The aphid species Aphis craccivora has been shown to be a vector for Alfalfa leaf curl virus, and the treehopper species Spissistilus festinus is the likely vector of Grapevine red blotch virus. In addition, two highly divergent groups of viruses found infecting citrus and mulberry plants have been assigned to the new species Citrus chlorotic dwarf associated virus and Mulberry mosaic dwarf associated virus, respectively. These species have been left unassigned to a genus by the ICTV because their particle morphology and insect vectors are unknown.

Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus.

The family Geminiviridae includes plant-infecting circular single-stranded DNA viruses that have geminate particle morphology. Members of this family infect both monocotyledonous and dicotyledonous plants and have a nearly global distribution. With the advent of new molecular tools and low-cost sequencing, there has been a significant increase in the discovery of new geminiviruses in various cultivated and non-cultivated plants. In this communication, we highlight the establishment of three new genera (Becurtovirus, Eragrovirus and Turncurtovirus) to accommodate various recently discovered geminiviruses that are highly divergent and, in some cases, have unique genome architectures. The genus Becurtovirus has two viral species, Beet curly top Iran virus (28 isolates; leafhopper vector Circulifer haematoceps) and Spinach curly top Arizona virus (1 isolate; unknown vector), whereas the genera Eragrovirus and Turncurtovirus each have a single assigned species: Eragrostis curvula streak virus (6 isolates; unknown vector) and Turnip curly top virus (20 isolates; leafhopper vector Circulifer haematoceps), respectively. Based on analysis of all of the genome sequences available in public databases for each of the three new genera, we provide guidelines and protocols for species and strain classification within these three new genera.

Revisiting the classification of curtoviruses based on genome-wide pairwise identity.

Members of the genus Curtovirus (family Geminiviridae) are important pathogens of many wild and cultivated plant species. Until recently, relatively few full curtovirus genomes have been characterised. However, with the 19 full genome sequences now available in public databases, we revisit the proposed curtovirus species and strain classification criteria. Using pairwise identities coupled with phylogenetic evidence, revised species and strain demarcation guidelines have been instituted. Specifically, we have established 77 % genome-wide pairwise identity as a species demarcation threshold and 94 % genome-wide pairwise identity as a strain demarcation threshold. Hence, whereas curtovirus sequences with >77 % genome-wide pairwise identity would be classified as belonging to the same species, those sharing >94 % identity would be classified as belonging to the same strain. We provide step-by-step guidelines to facilitate the classification of newly discovered curtovirus full genome sequences and a set of defined criteria for naming new species and strains. The revision yields three curtovirus species: Beet curly top virus (BCTV), Spinach severe surly top virus (SpSCTV) and Horseradish curly top virus (HrCTV).

Analysis of the full-length genome sequence of papaya lethal yellowing virus (PLYV), determined by deep sequencing, confirms its classification in the genus Sobemovirus.

Papaya lethal yellowing virus (PLYV) causes an economically important disease in papayas in northeastern Brazil. Based on biological and molecular properties, PLYV has been tentatively assigned to the genus Sobemovirus. We report the sequence of the full-length genome of a PLYV isolate from Brazil, determined by deep sequencing. The PLYV genome is 4,145 nt long and contains four ORFs, with an arrangement identical to that of sobemoviruses. The polyprotein and CP display significant sequence identity with the corresponding proteins of other sobemoviruses. Pairwise comparisons and phylogenetic analysis based on complete nucleotide sequences confirm the classification of PLYV in the genus Sobemovirus.

Six novel begomoviruses infecting tomato and associated weeds in Southeastern Brazil.

The incidence of tomato-infecting begomoviruses has sharply increased in Brazil following the introduction of the B biotype of the whitefly vector in the early 1990s. Five definitive species and six tentative species have been described since then. Here, we report the detection of members of an additional six novel species, three in tomato and three infecting weeds that are commonly associated with tomato fields: Blainvillea rhomboidea, Sida rhombifolia and Sida micrantha. Tomato and weed samples were collected in two major tomato-growing regions of southeastern Brazil in 2005 and 2007. Two of the novel viruses were present in tomato plants collected in Paty do Alferes, Rio de Janeiro state. Three novel viruses were present in weed samples collected in Coimbra, Minas Gerais state. One virus was present in tomato samples collected at both locations. Genome features indicate that all six species are typical New World, bipartite begomoviruses. However, the viruses belonging to two of the novel species did not cluster with the Brazilian viruses in a phylogenetic tree. These species could represent a distinct lineage of New World begomoviruses, found in Brazil for the first time.

Cowpea aphid-borne mosaic virus (CABMV) is widespread in passionfruit in Brazil and causes passionfruit woodiness disease.

Leaf samples of yellow passionfruit (Passiflora edulis f. flavicarpa) displaying fruit woodiness symptoms were collected in seven Brazilian states and the Federal District. Viral infection was confirmed by host range and ELISA, and fourteen viral isolates were obtained. All isolates were capable of infecting several leguminous host species, although differences in symptom severity were noticeable. Woodiness symptoms were reproduced in yellow passionfruit, and mosaic symptoms were induced in common bean. All isolates infected cowpea, reported as a non-host of passion fruit woodiness virus (PWV). Indirect ELISA demonstrated that all isolates were serologically related to each other and also to cowpea aphid-borne mosaic virus (CABMV). The complete sequence of the capsid protein was determined for all isolates. Comparison of these sequences with those of other potyviruses indicated the highest identity with CABMV isolates (85 to 94%). Identity with PWV isolates ranged from 54 to 70%. Phylogenetic analysis grouped all of the Brazilian isolates in a monophyletic cluster with the CABMV isolates, clearly distinct from the PWV isolates. Furthermore, this analysis demonstrated that a group of previously characterized isolates from Brazil that had been designated as PWV should be reclassified as CABMV. Together, these results provide unequivocal evidence that, in Brazil, passionfruit woodiness disease is primarily caused by CABMV. The presence of PWV in Brazil has yet to be confirmed.