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Transcriptome datasets available in public domain serve as valuable resource for identification and characterization of novel viral genomes. Poleroviruses are economically important plant-infecting RNA viruses belonging to the family Luteoviridae. In the present study, we explored the plant transcriptomes available in public domain and identified five putative novel poleroviruses tentatively named as Foeniculum vulgare polerovirus (FvPV), Kalanchoe marnieriana polerovirus (KmPV), Paspalum notatum polerovirus (PnPV), Piper methysticum polerovirus (PmPV), Trachyspermum ammi polerovirus (TaPV) and a novel enamovirus named as Celmisia lyallii enamovirus (ClEV) in Foeniculum vulgare, Kalanchoe marnieriana, Paspalum notatum, Piper methysticum, Trachyspermum ammi and Celmisia lyallii, respectively. Coding-complete genomes (5.56−5.74 kb) of CIEV, KmPV, PnPV, PmPV and TaPV were recovered while only the partial genome of FvPV could be recovered. The genome organization of identified viruses except ClEV is 5’−P0−P1−P2−P3a−P3−P4−P5−3’ while that of ClEV is 5’−P0−P1−P2−P3− P5−3’. Phylogenetic analysis and genome organization revealed the relatedness of FvPV, KmPV, PnPV, PmPV and TaPV to other poleroviruses and ClEV to other enamoviruses.

Arracacha is a root crop cultivated in several countries of South America. The commercial product, which is the storage root, has multiple nutritional properties: its fine starch makes it easily digestible, and it contains high levels of calcium and vitamin A. High throughput sequencing (HTS) technology has been applied successfully for virus discovery in different agricultural crops, and it has been proposed to apply it in routine pathogen detection. Using HTS, novel sequences related to crinivirus and vitivirus were identified in apparently symptomeless arracacha, which were assembled into contigs located in different positions of the genome. Based on those sequences’ primers were designed to amplify corresponding sequences from further arracacha accessions and potato plant samples collected from farmers’ fields in Colombia and Peru. It was possible to determine the near complete genomes of these viruses. In addition, sequences related to an enamovirus and ST9-like RNA were also identified in arracacha plants using HTS.

Pea (Pisum sativum L.) is an economically important legume crop that is commonly used as dry beans, fresh peas, pods and shoots (Guo et al. 2009). Pea enation mosaic is an important virus disease of pea caused by two viruses in an obligate symbiosis, pea enation mosaic virus 1 (PEMV-1, Enamovirus, Luteoviridae) and pea enation mosaic virus 2 (PEMV-2, Umbravirus, Tombusviridae) (Hema et al. 2014). In November 2019, foliar yellow mosaic and vein enations symptoms were observed from pea plants in five fields of Honghe autonomous prefecture, Yunnan province, China. Incidence of symptomatic plants ranged from 20 to 40% and was distributed in both small and large fields. Leaves with typical virus-like symptoms were collected from five symptomatic pea plants in two fields and used for total RNA extraction. The five extracts of equimolar quantities were pooled into a sample and subjected to High Throughput Sequencing (HTS) by Illumina HiSeq system. Analyses of raw RNA reads were performed using CLC Genomics Workbench 12 (Qiagen). A total of 60,009,746 RNA reads were obtained from the sample, and de novo assembly of the reads using the CLC Genomics generated 88,105 contigs. BLASTN searches revealed the presence of contigs with high similarities to PEMV-1, PEMV-2, Pea seed-borne mosaic virus, and Bean yellow mosaic virus. To confirm the presence of PEMV-1 and PEMV-2 in the samples, two virus-specific primer pairs were designed based on the contig sequences obtained by HTS in this study. Primer pairs PEMV-1F/PEMV-1R (5'-ATGCCGACTAGATCGAAATC-3'/5'-TCAGAGGGAGGCATTCATTA-3') that flank the cp gene of PEMV-1 and PEMV-2F/PEMV-2R (5'-ATGACGATAATCATTAATG-3'/5'-TCACCCGTAGTGAGAGGCA-3') that target the ORF3 region of PEMV-2 were used to amplify the two viruses in RT-PCR. DNA fragments of the expected sizes (PEMV-1, 570 bp; PEMV-2, 693 bp) were amplified from all five samples. The RT-PCR products were cloned and sequenced. Sequence analysis showed that the 570-bp amplicon (MT481989) shared the highest nucleotide sequence identity of 98.95% with PEMV-1 (Z48507), while the 693-bp fragment (MT481990) had the highest nucleotide sequence identity of 97.4% with PEMV-2 isolate JKI (MK948534). One gram of the symptomatic leaves from each of the five plants was homogenized with 5 mL of 0.01 M phosphate-buffered saline (PBS buffer), pH 7.0. Each of the resulted saps was used to inoculate onto five healthy pea seedlings. A total of 25 healthy pea seedlings were inoculated, and 16 inoculated plants developed yellowing and mottling at 10 days post inoculation (dpi); no symptoms were observed on control plants inoculated only with PBS buffer. The formation of the typical enation was observed along the veins of lower side of the symptomatic leaves of the inoculated plants at 30 dpi. PEMV-1 and PEMV-2 infection were confirmed by RT-PCR assays using the specific primer pairs described above. Although the presence of the pea enation mosaic virus complex was suspected in China based on symptomatology (Brunt et al. 1997), to our knowledge, this is the first molecular confirmation of PEMV-1 and PEMV-2 occurrence in China. The co-infection of PEMV-1 and PEMV-2 usually cause severe yield losses; therefore, integration of detection and control measures is important in pea production regions where the two viruses occurred.

Alfalfa dwarf disease, probably caused by synergistic interactions of mixed virus infections, is a major and emergent disease that threatens alfalfa production in Argentina. Deep sequencing of diseased alfalfa plant samples from the central region of Argentina resulted in the identification of a new virus genome resembling enamoviruses in sequence and genome structure. Phylogenetic analysis suggests that it is a new member of the genus Enamovirus, family Luteoviridae. The virus is tentatively named "alfalfa enamovirus 1" (AEV-1). The availability of the AEV-1 genome sequence will make it possible to assess the genetic variability of this virus and to construct an infectious clone to investigate its role in alfalfa dwarfism disease.

Here, we report the identification and characterization of two novel viruses associated with bird's-foot trefoil. Virus sequences related to those of enamoviruses (ssRNA (+); Luteoviridae; Enamovirus) and nucleorhabdoviruses (ssRNA (-); Rhabdoviridae; Nucleorhabdovirus) were detected in Lotus corniculatus transcriptome data. The genome of the tentatively named "bird's-foot trefoil-associated virus 1" (BFTV-1) is a 13,626-nt-long negative-sense ssRNA. BFTV-1 encodes six predicted gene products in the antigenome orientation in the canonical order 3'-N-P-P3-M-G-L-5'. The genome of the proposed "bird's-foot trefoil-associated virus 2" (BFTV-2) is 5,736 nt long with a typical 5΄-PO-P1-2-IGS-P3-P5-3' enamovirus genome structure. Phylogenetic analysis indicated that BFTV-1 is closely related to datura yellow vein nucleorhabdovirus and that BFTV-2 clusters into a monophyletic lineage of legume-associated enamoviruses. This subclade of highly related and co-divergent legume-associated viruses provides insights into the evolutionary history of the enamoviruses.

Bird’s-foot trefoil (Lotus corniculatus) is a nutritious forage crop, employed for livestock foraging around the world. Here, we report the identification and characterization of two novel viruses associated with bird’s-foot trefoil. Virus sequences with affinity to enamoviruses (ssRNA (+);Luteoviridae; Enamovirus) and nucleorhabdoviruses (ssRNA (-);Rhabdoviridae; Nucleorhabdovirus) were detected inL. corniculatustranscriptome data. The tentatively named birds-foot trefoil associated virus 1 (BFTV-1) genome organization is characterized by 13,626 nt long negative-sense ssRNA. BFTV-1 presents in its antigenome orientation six predicted gene products in the canonical order 3′-N-P-P3-M-G-L-5′. The proposed birds-foot trefoil associated virus 2 (BFTV-2) 5,736 nt virus sequence presents a typical 5′-PO-P1-2-IGS-P3-P5-3′ enamovirus genome structure. Phylogenetic analysis suggests that BFTV-1 is closely related toDatura yellow vein nucleorhabdovirus, and that BFTV-2 clusters into a monophyletic cluster of legumes-associated enamoviruses. This sub-clade of highly related and co-divergent legume associated viruses provides insights on the evolutionary history of the enamoviruses. The bioinformatic reanalysis of SRA libraries deposited in the NCBI database constitutes an emerging approach to the discovery of novel plant viruses which should be important for both quarantine purposes and disease management.

The complete nucleotide sequence of an isolate of Citrus vein enation virus (CVEV-XZG) from China has been determined for the first time. The genome consisted of 5 983 nucleotides, coding for five open reading frames (ORFs), had a similar genomic organization features with Pea enation mosaic virus (PEMV). Nucleotide and deduced amino acid sequence identity of the five ORFs compared to isolate CVEV VE-1 range from 97.1 to 99.0% and 97.4 to 100.0%, these values compared to isolate PEMV-1 range from 45.2 to 51.6% and 31.1 to 45.2%. Phylogenetic analysis based on the complete genome sequence showed that the isolate CVEV-XZG had close relationship with Pea enation mosaic virus. The results supports CVEV may be a new member of genus Enamovirus. The full sequence of CVEV-XZG presented here may serve as a basis for future study of CVEV in China.

We report the complete genome sequence of a viral pathogen detected in alfalfa samples from Sudan and provisionally named alfalfa enamovirus 2 (AEV-2). Based on high nucleotide and amino acid identities, AEV-2 represents a strain of a newly discovered alfalfa enamovirus 1 that has only been described in Argentina.

In this study, we describe a novel putative Enamovirus member, Grapevine enamovirus-1 (GEV-1), discovered by high-throughput sequencing (HTS). A limited survey using HTS of 17 grapevines (Vitis spp.) from the south, southeast, and northeast regions of Brazil led to the detection of GEV-1 exclusively on southern plants, infecting four grapevine cultivars (Cabernet Sauvignon, Semillon, CG 90450, and Cabernet franc) with a remark-able identity of around 99% at the nucleotide level. This novel virus was only detected in multiple-virus infected plants exhibiting viral-like symptoms. GEV-1 was also detected on a cv. Malvasia Longa by RT-PCR. We performed graft-transmissibility assays on GEV-1. The organization, products, and cis-acting regulatory elements of GEV-1 genome are also discussed here. The near complete genome sequence of GEV-1 was obtained during the course of this study, lacking only part of the 3 0 untranslated terminal region. This is the first report of a virus in the family Luteoviridae infecting grapevines. Based on its genomic properties and phylogenetic analyses, GEV-1 should be classified as a new member of the genus Enamovirus . Keywords High-throughput sequencing Virus discovery Luteoviridae Grapevine enamovirus-1 GEV-1

An antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA) method was developed and validated for the detection of Bean leafroll virus (BLRV) and Pea enation mosaic virus (PEMV), two of the important viral pathogens of several legume crops. The coat protein (CP) gene of each of the viruses was bacterially expressed as a fusion protein containing an N-terminal hexa-histidine tag and used as an antigen to produce antisera in rabbits. The antiserum to BLRV could detect the virus in leaf samples in up to 1:1000 dilution, and the PEMV antiserum detected the homologous virus in leaf samples of dilutions up to 1:6400. No serological cross-reactivity was observed between anti-BLRV and anti-PEMV sera. The ACP-ELISA assays were then used for estimating the prevalence of these two viruses in alfalfa, pea and vetch over a three-state area in the US Pacific Northwest over a 2-year period and virus incidence was mapped. Availability of rapid and sensitive ELISA assays facilitate virus disease mapping efforts and screening germplasm for virus resistance.

The family Luteoviridae comprises of three genera Luteovirus, Enamovirus and Polerovirus, of which, only two genera Luteovirus and Polerovirus are known in India. Though luteovirus and polerovirus infects many crops throughout the world, only a few have been documented in India. Luteoviruses were reported in field crops like barley (barley yellow dwarf virus) and chickpea (chickpea stunt virus), while polerovirus was recorded from potato, jute (potato leaf roll virus [PLRV]) sugarcane (sugarcane yellow leaf virus [SCYLV]) and cotton (cotton leaf roll dwarf virus) crops from India. SCYLV and PLRV are the most important poleroviruses as they are of serious constraints in all the sugarcane and potato growing states of India. SCYLV infection causes 39–43% reductions in plant growth and 30–34% loss in yield in sugarcane. The virus is mainly transmitted through infected seed canes. The secondary spread of the virus in the field is by the aphid vectors. PLRV is reported to cause 50–60% yield losses in potato crop and this virus is tuber borne and transmitted mainly by aphid vectors in a circulative non-propagative manner. The complete genome sequences SCYLV and PLRV isolates from sugarcane and potato are available from India. This book chapter deals with an uptodate information available on distribution, biological properties, identification, serological relationships, genetic diversity and transmission of the luteoviruses and poleroviruses reported on different crops from India.

Pea enation mosaic virus (PEMV) infects several legume crops, including chickpea (Cicer arietinum), faba bean (Vicia faba), lentil (Lens culinaris) and pea (Pisum sativum). The virus caused yield losses of food legumes in the Pacific Northwestern US during 1983, 1987 and 1990 [1]. Our recent surveys of pea and alfalfa fields in the states of Washington and Idaho, USA, have shown the prevalence of PEMV on pea. PEMV consists of a large (RNA-1 or PEMV-1) and a small (RNA-2 or PEMV-2) single-stranded positive-sense RNA, which are encapsidated separately into distinct isometric particles [2]. PEMV-2 is one of the seven distinct virus species in the genus Umbravirus. The other species of this genus are Carrot mottle virus (CMoV), Carrot mottle mimic virus (CMoMV), Groundnut rosette virus (GRV), Lettuce speckles mottle virus (LSMV), Tobacco mottle virus (TMoV) and Tobacco bushy top virus (TBTV) [3]. Members of this group lack the coat protein (CP) gene in their genomes and depend on a helper virus for survival [4]. PEMV-2 in association with PEMV-1 (genus Enamovirus), form a symbiotic bipartite virus complex referred to as PEMV. It has been suggested that RNA-1 and RNA-2 are essential for PEMV infection [4, 5]. The RNA-1 (5706 nucleotides) encodes five major ORFs (1-5) that have nucleotide and amino acid sequence similarities with subgroup II luteoviruses [5]. While PEMV-1 provides the necessary encapsidation and vector-transmission abilities for PEMV-2, the latter provides PEMV-1 with long-distance movement and mechanical transmission functions. As part of an ongoing project to determine the genetic diversity of PEMV, we recently characterized the genome of PEMV-1 from the Pacific Northwest [6]. So far, there are only two complete genomic sequences of PEMV-2 in GenBank (NC_003853 and AY714213). To better understand sequence diversity of PEMV-2, the genome structure and organization of two PEMV isolates, one from Idaho (PEMV-2-ID) and one from Washington (PEMV-2-WA), were determined in this study. The PEMV-2 genome (*4.2 kbp), like that of other umbraviruses, predominantly consists of four ORFs (1-4), which perform diverse functions [4, 7, 8]. RNA-2 lacks a polyadenylation signal at its 30 end and contains a large 50 genome-linked protein [9]. ORF-1, at the 50 end of the virus genome, initiates after a short, 20-nt non-coding region (NCR) and encodes a putative 33-kDa protein of unknown function. ORF-2 overlaps with ORF-1 at its 30 end and potentially encodes a protein of 65 kDa through a frameshift mechanism. ORF-2 contains sequence motifs characteristic of a viral RNA-dependent RNA-polymerase (RdRp). The presence of a polymerase cassette in RNA-2 also reveals its independent replicative capabilities. Also, due to the presence of an octanucleotide frameshift signal ‘‘GGATTTTT’’ immediately upstream of the stop codon of ORF-1, ORF-1, along with ORF-2, is expressed by a -1 frameshift mechanism as a minor translation product of 97 kDa [4]. ORFs 3 and 4 in the genome occur after a nonThe sequences described here were deposited in the GenBank database with the following accession numbers: JF713435 for PEMV2-WA and JF713436 for PEMV-2-ID.

This study, using RT-PCR, is the first comprehensive assessment since 1991 of a generic detection method for the Luteoviridae. Thirteen Luteoviridae species were detected using three separate sets of low-degeneracy generic primers with RT-PCR to amplify 68-, 75- and 129/156-bp regions of the Luteoviridae coat-protein gene. Species detected include all members of the genus Luteovirus [Barley yellow dwarf virus (BYDV)-PAV, BYDV-PAS, BYDV-MAV (129 and/or 156 bp amplicons), Soybean dwarf virus, Bean leafroll virus (68 bp amplicon)] and eight of nine species from the genus Polerovirus [Beet western yellows virus, Beet chlorosis virus, Beet mild yellowing virus, Turnip yellows virus, Potato leafroll virus, Cucurbit aphid-borne yellows virus, Cereal yellow dwarf virus-RPV (68-bp amplicon) and Sugarcane yellow leaf virus (75-bp amplicon)]. These primers were not able to detect Carrot red leaf virus, Sweet potato leaf speckling virus (both belong to unassigned Luteoviridae) and Pea enation mosaic virus-1 (genus Enamovirus). A synthetic positive control containing all primer sequence priming sites was designed to facilitate this method as a generic tool for use with a variety of host plants. The Luteoviridae primers described in this study present a simple infection-detection tool of benefit to biosecurity authorities in nursery-stock surveillance, disease management or outbreak prevention, and may also be useful in detection of as-yet undiscovered species within the Luteovirus and Polerovirus genera.

In January 2016, 35 leaf dried samples of chilli pepper (Capsicum sp.) were submitted to Fera Science Ltd, from six districts in Rwanda: Ruhango, Nyanza, Rulindo, Kayonza, Nyagatare and Kirehe. The samples were sent in following the …

The family Luteoviridae consists of eight viruses assigned to three different genera, Luteovirus, Polerovirus and Enamovirus. The complete genomic sequences of pea enation mosaic virus (genus Enamovirus) and bean leafroll virus (genus Luteovirus) from the Pacific Northwest, USA, were determined. Annotation, sequence comparisons, and phylogenetic analysis of selected genes together with those of known polero- and enamoviruses were conducted.

Pea enation mosaic disease is caused by an obligatory association between the enamovirus Pea enation mosaic virus-1 (PEMV-1) and the umbravirus Pea enation mosaic virus-2 (PEMV-2). Encapsidated RNAs 1 and 2 are covalently linked to a 3138 Da VPg encoded by the RNA of PEMV-1. To determine the role of the VPg in the pathogenicity of PEMV (PEMV-1+PEMV-2), the infectivity of clones with mutations in key amino acids in the VPg was evaluated in protoplasts and in plants. Using quantitative, real-time RT-PCR, we concluded that the inability of certain mutants to infect plants was due to their replicative (and not their movement) incompetence. Mutant clones that produced delayed and less severe infections accumulated 10- to 100-fold less RNA-1 compared to WT-RNA-1 both in plants and in protoplasts. The RNAs of clones that produced WT-like infections accumulated to levels similar to those of WT-PEMV. Also, we demonstrate that the severity of symptoms produced by WT-PEMV is proportional to the amount of RNA-1 that accumulates in infected plants and seems to be independent of the amount of RNA-2. A dual role for the VPg in the pathogenicity of PEMV is proposed.

We have derived the genomic nucleotide sequence of an emerging virus, the Sugarcane yellow leaf virus (ScYLV), and shown that it produces one to two subgenomic RNAs. The family Luteoviridae currently includes the Luteovirus, Polerovirus, and Enamovirus genera. With the new ScYLV nucleotide sequence and existing Luteoviridae sequence information, we have utilized new phylogenetic and evolutionary methodologies to identify homologous regions of Luteoviridae genomes, which have statistically significant altered nucleotide substitution ratios and have produced a reconstructed phylogeny of the Luteoviridae. The data indicate that Pea enation mosaic virus-1 (PEMV-1), Soybean dwarf virus (SbDV), and ScYLV exhibit spatial phylogenetic variation (SPV) consistent with recombination events that have occurred between poleroviral and luteoviral ancestors, after the divergence of these two progenitor groups. The reconstructed phylogeny confirms a contention that a continuum in the derived sequence evolution of the Luteoviridae has been established by intrafamilial as well as extrafamilial RNA recombination and expands the database of recombinant Luteoviridae genomes that are currently needed to resolve better defined means for generic discrimination in the Luteoviridae (D'Arcy, C. J. and Mayo, M. 1997. Arch. Virol. 142, 1285-1287). The analyses of the nucleotide substitution ratios from a nucleotide alignment of Luteoviridae genomes substantiates the hypothesis that hot spots for RNA recombination in this virus family are associated with the known sites for the transcription of subgenomic RNAs (Miller et al. 1995. Crit. Rev. Plant Sci. 14, 179-211), and provides new information that might be utilized to better design more effective means to generate transgene-mediated host resistance.

Pea enation mosaic virus (PEMV) is a bipartite virus, transmitted mechanically and by aphids in the circulative manner. Virus capsid proteins, coded by RNA-1 are involved in aphid transmissibility. Using an antiserum against a European (UK) aphid-transmissible isolate, the capsid proteins of six PEMV isolates, each aphid-transmissible or non-transmissible, were compared using Western blots. All isolates have a major coat protein of approximately 22 kDa, but also additional proteins. RNA-1 complementary DNAs corresponding to ORFs 4 and 5 of isolate SP were cloned and sequenced. This had 95% homology at the nucleotide level and a 96% homology at the amino acid level with the sequence of a North American isolate. Most amino acid differences were in the region corresponding to ORF 5, some of which could result in potential changes in the conformation of the protein. This is the first report on the sequence of an European PEMV isolate and the first direct comparison of viral capsid complexity in different PEMV isolates. Zusammenfassung Vergleich von Isolaten des Erbsenenationenmosaikvirus (PEMV): Sequenz der Hullproteingene und der fur die Blattlausubertragungsproteine codierenden Gene eines europaischen Isolates Das Erbsenenationenmosaikvirus (PEMV) ist ein zweigeteiltes Virus, das mechanisch und durch Blattlause zirkulativ ubertragen wird. Von RNA-1 codierte Hullproteine des Virus spielen fur die Blattlausubertragbarkeit eine Rolle. Mit einem Antiserum gegen ein blattlausubertragbares Isolate aus dem Vereinigten Konigreich wurden die Hullproteine von sechs blattlausubertragbaren bzw. nicht ubertragbaren PEMV-Isolaten in Western blots verglichen. Alle Isolate besitzen ein groses Hullprotein von ca. 22 kDa, aber auserdem weitere Proteine. RNA-1 cDNAs, die mit den ORFs 4 und 5 des Isolats SP korresponierten, wurden kloniert und sequenziert. Auf Nukleotidebene wurde eine Homologie von 95% mit einem nordamerkanischen Isolat festgestellt, auf Aminosaureebene eine Homologie von 96%. Die meisten Unterschiede bei den Aminosauren fanden sich in der ORF 5 entsprechenden Region, einige von ihnen sind moglicherweise fur eine Konformationsanderung des Proteins verantwortlich. Dies ist der erste Bericht uber die Sequenz eines europaischen PEMV-Isolats und der erste direkte Vergleich der Komplexitat von Virushullen verschiedener PEMV-Isolate.

Pea (Pisum sativum L.) plants were transformed in planta by injection/electroporation of axillary meristems with a chimeric pea enation mosaic virus (PEMV) coat protein gene contruct. R1 progenies of these plants were shown to harbor the transgene by polymerase chain reaction (PCR) and genomic Southern analysis, while transgene expression was demonstrated by western blot analysis. Transgenic R2, R3 and R4 plants displayed delayed or transient PEMV multiplication and attenuated symptoms as compated to control inoculated individuals.

Luteoviruses and the luteovirus-like pea enation mosaic virus (PEMV; genus Enamovirus) are transmitted by aphids in a circulative, nonreplicative manner. Acquired virus particles persist for several weeks in the aphid hemolymph, in which a GroEL homolog, produced by the primary endosymbiont of the aphid, is abundantly present. Six subgroup II luteoviruses and PEMV displayed a specific but differential affinity for Escherichia coli GroEL and GroEL homologs isolated from the endosymbiotic bacteria of both vector and nonvector aphid species. These observations suggest that the basic virus-binding capacity resides in a conserved region of the GroEL molecule, although other GroEL domains may influence the efficiency of binding. Purified luteovirus and enamovirus particles contain a major 22-kDa coat protein (CP) and lesser amounts of an approximately 54-kDa readthrough protein, expressed by translational readthrough of the CP into the adjacent open reading frame. Beet western yellows luteovirus (BWYV) mutants devoid of the readthrough domain (RTD) did not bind to Buchnera GroEL, demonstrating that the RTD (and not the highly conserved CP) contains the determinants for GroEL binding. In vivo studies showed that virions of these BWYV mutants were significantly less persistent in the aphid hemolymph than were virions containing the readthrough protein. These data suggest that the Buchnera GroEL-RTD interaction protects the virus from rapid degradation in the aphid. Sequence comparison analysis of the RTDs of different luteoviruses and PEMV identified conserved residues potentially important in the interaction with Buchnera GroEL.

Deletions in the coat protein (CP) open reading frame (ORF)-3 of pea enation mosaic enamovirus (PEMV) did not inhibit the systemic movement of PEMV. As early as 48 h postinoculation, truncated viral RNAs were detected in the roots and uninoculated leaves of infected plants. The detected concentrations of truncated RNAs varied significantly between experiments but were similar to those extracted from plants infected with wild-type (WT)-PEMV. Pea plants infected with CP-less mutants exhibited wilting and necrosis that appeared faster and were more severe than observed in infections with WT-PEMV. Moreover, the absence of the CP-ORF and its protein product did not affect the formation of either double-membrane bound vesicles or replication complexes characteristic of infections by PEMV. The potential role of these structures as the movement vehicles of the infectious entity of PEMV in plants is discussed.

Pea enation mosaic virus (PEMV) is composed of two autonomously replicating virus RNAs related to the genomic RNAs of viruses in the genera Luteovirus and Umbravirus. The transmission of PEMV resembles that of its luteovirus relatives in utilizing circulative aphid transmission. However, unlike its luteovirus counterparts, PEMV can also be mechanically transmitted. Prolonged mechanical passage of PEMV can lead to the loss of aphid transmissibility, a trait that is mirrored by specific changes in the PEMV virion composition. These changes were used to examine the virus contribution to vector transmission and the mechanisms by which it is regulated. Using a local lesion isolation technique, one aphid transmissible and two aphid non-transmissible isolates of PEMV were compared. Structural analysis of a 54 kDa minor structural subunit unique to the aphid transmissible isolate demonstrated that it was a fusion of the 21 kDa virus coat protein and a 33 kDa protein encoded immediately downstream of the 21 kDa ORF, consistent with the formation of the 54 kDa subunit by translational readthrough. Genetic analyses utilizing exchanges between infectious in vitro transcripts of each isolate demonstrated that although the 33 kDa protein was non-essential for infection, its presence was mandatory for aphid transmission, and that specific changes within the 33 kDa ORF were sufficient to confer or abolish aphid transmission. This study also demonstrates that isolates of PEMV exist as mixtures of aphid transmissible and non-transmissible genotypes, and provides insight into the mechanisms used by this virus to down-regulate aphid transmission in response to a specific selection pressure.

Understanding the molecular mechanisms involved in plant virus–vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus, Luteoviridae) and Pea enation mosaic virus 2 (PEMV2, Umbravirus, Tombusviridae) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum, and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum. Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

The genome of pea enation mosaic virus (PEMV) is composed of two taxonomically unrelated RNAs, interacting to create what has traditionally been considered a bipartite virus. The cohesiveness of this interaction was assessed by examining the autonomy of each RNA in viral replication, coat protein expression and systemic invasion. Using a pea protoplast system, in vitro transcripts of RNA1 were found to be capable of initiating RNA2-independent replication, including the formation of the distinctive nuclear membrane-based replication complex associated with wild-type PEMV infection. Western blotting and electron microscopic analysis demonstrated that the synthesis of the RNA1-encoded coat protein, as well as virion assembly, was also independent of RNA2-directed functions. Mechanical inoculations with transcripts of RNA1 failed to establish a systemic RNA1 infection, whereas inoculations with RNA2 were able to establish a largely asymptomatic systemic infection. Combined inoculum containing RNA1 and RNA2 transcripts were able to recreate wild-type PEMV symptomatology, demonstrating the dependence of RNA1 on RNA2 for mechanical passage. With the notable exception of the adaptation of PEMV to establish a true systemic invasion, these data further strengthen the analogy between PEMV and the helper-dependent complexes associated with members of the luteovirus group.

Publisher Summary The genome of viruses in the family Luteoviridae consists of a single positivesense linear RNA molecule of about 5.7 kilobases (kb) packaged in small icosahedral virions measuring 25 nm in diameter. The family Luteoviridae is now divided into three genera: Luteovirus, type species barley yellow dwarf virus-PAV (BYDV-R\V); Polerovirus, type species potato leafroll virus (PLRV), and Enamovirus, type species pea enation mosaic virus-1 (PEMV-1) [7]. The genomic RNAs of all three genera contain five or six main open reading frames (ORFs) arranged in two clusters. In the 5'-proximal cluster, ORFS 1 and 2 encode the viral RNA-dependent RNA polymerase and display distinct taxonomic affinities. Luteovirus polymerase is Carmovirus-like, whereas Polerovirus and Enamovirus polymerases are sobemovirus-like. Poleroviruses and PEMV-1 possess a 5"-terminal genome-linked protein (VPg) derived from ORE 1, whereas luteoviruses do not. Poleroviruses and PEMV-1 also contain a 5"-proximal ORE 0 of unknown frinction, which is not present in luteoviruses. The genes in the 3'-proximal cluster are translated from a subgenomic RNA.

The 5895 nucleotide long single-stranded RNA genome of Sugarcane yellow leaf virus Florida isolate (SCYLV-F) includes six major ORFs. All but the first of these are homologous to genes of known function encoded by viruses of the three newly defined genera in the Luteoviridae (‘luteovirids’), i.e. poleroviruses, luccccteoviruses and the enamoviruses. SCYLV-F ORFs 1 and 2 are most closely related to their polerovirus counterparts, whereas SCYLV-F ORFs 3 and 4 are most closely related to counterparts in luteovirus genomes, and SCYLV-F ORF5 is most closely related to the read-through protein gene of the only known enamovirus. These differences in affinity result from inter-species recombination. Two recombination sites in the genome of SCYLV-F map to the same genomic locations as previously described recombinations involving other luteovirids. A fourth type of luteovirid, Soybean dwarf virus, has already been described. Our analyses indicate that SCYLV-F represents a distinct fifth type.

Among the plant viruses, those in the family Luteoviridae (luteovirus is used here to refer to all definitive and tentative members of this family) are some of the most widespread and economically important (Miller 1994). They cause important diseases worldwide in nearly all of the crops human beings grow for food and fiber (Dueeus 1977; Miller 1994). They are obligately vector transmitted from plant to plant by specific aphids, and, once acquired, a vector aphid may carry a corresponding luteovirus for the remainder of the aphid’s life; the virus:vector transmission relationship is circulative:nonpropagative (Gildow 1987). Within the plant host the luteovirus infection is limited to the phloem tissues, and luteoviruses are generally classified as nonmechanically transmissible (D’Arcy et al. 1998).

Of the 47 genera of plant viruses recognized by the International Committee on Taxonomy of Viruses (Murphy et al., 1995), 33 have genomes composed of single-stranded, positive-sense RNA. Of these 33 genera, 9 have bipartite genomes, 19 have isometric particles, and 6 have both characteristics. This volume therefore deals with an important fraction of the known types of plant viruses, namely, members of the genera Comovirus, Dianthovirus, Enamovirus, Fabavirus, Idaeovirus, and Nepovirus.

Pea enation mosaic enamovirus (PEMV) has long been recognized to have a unique combination of properties and has been assigned to a monotypic group (Harrison et al., 1971), now given the generic name Enamovirus (de Zoeten and Demler, 1995). The generic characters include a bipartite, plussense, single-stranded RNA (ssRNA) genome, both RNA species (RNA-1 and RNA-2) being needed to establish a systemic infection and each being encapsidated in separate morphologically distinct icosahedral particles. The virus is transmissible by aphids in the persistent (circulative) manner and by mechanical inoculation. Some isolates also contain a nonessential third RNA species, designated RNA-3.

Pea enation mosaic enamovirus (PEMV) is important economically and very interesting scientifically. The disease it induces in peas was first discovered in New York State by Osborn (1935), who showed that the causal virus is aphid-transmitted in a persistent manner. The disease was named “pea enation mosaic” by Stubbs (1937), who described the symptoms in different varieties of pea (Pisum sativum), host range, insect transmission, temperature relations, and physical properties. Since these pioneering researches, many studies have been made on diverse scientific aspects of PEMV and the disease it incites. This chapter will present current knowledge on the ecology of PEMV and on the control of the pea enation mosaic disease.

Luteoviridae is a plant virus family of 26 species which causes yield losses in cereals potatoes and other economically important crops worldwide Accurate detection is an important component of controlling the spread of luteoviruses and reducing yield losses PCRbased detection is often the method of choice for Luteoviridae as it is more sensitive than serological methods which frequently fail to detect infection due to the low concentration of luteoviruses in plants Since the currently available luteovirus primers are mostly speciesspecific and work under different PCR conditions universal primers are desirable Seven primers were tested that were designed to target the most conserved regions of the Luteoviridae genomes and that possess high homology to over 75 of Luteoviridae species Thirty luteovirus isolates representing 15 species were obtained from New Zealand and overseas these included all five species from the Luteovirus genus eight of the nine species from the Polerovirus genus (except CYDV RPS) PEMV1 (the only species from the Enamovirus genus) and CtRLV (which is not assigned to a genus) Between them the three combinations of primers detected all 13 of the Luteovirus and Polerovirus species tested