ICTV Virus Taxonomy Profile: Nanoviridae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

Viral infection can reduce the net nitrogen inputs of legume break crops and cover crops.

Legumes are used in crop rotations by both large-scale and smallholder farmers alike to increase soil fertility, especially before high-nitrogen-demanding crops such as corn (maize). Legume crop residues and green manures are rich in nitrogen due to mutualistic rhizobia, bacteria that live in their roots and convert atmospheric nitrogen into a biologically available form. Growers can obtain recommendations from local extension offices about how much less inorganic nitrogen fertilizer needs to be added to a subsequent crop following different legume break crops for the predominant soil type (the nitrogen fertilizer replacement value, or NFRV). Due to the intimate relationship between legumes and rhizobia, conditions that affect plant health can also affect the rhizobia and how much nitrogen they provide. We use a combination of empirical data and previously published values to estimate reductions in nitrogen inputs under outbreaks of plant viruses of varying severity. We also use historical fertilizer prices to examine the economic impacts of this lost fertilizer for farmers. We find that fertilizer losses are greatest for crops that fix large amounts of nitrogen, such as clover and alfalfa as opposed to common bean. The economic impact on farmers is controlled by the proportion of plants with viral infections and the price of synthetic fertilizer. In a year of high disease prevalence, attention is normally focused on the yield of the diseased crops. We suggest that farmers growing legumes as break crops should be concerned about yields of subsequent crops as well. Viral diseases can be difficult to diagnose in the field, so the easiest way for farmers to prevent unexpected yield losses in subsequent crops is to test their soil when it is feasible to do so.

Biology and genetic diversity of phasey bean mild yellows virus, a common virus in legumes in Australia.

This study examined the natural and experimental host range and aphid and graft transmission of the tentative polerovirus phasey bean mild yellows virus (PBMYV). Eleven complete coding sequences from PBMYV isolates were determined from a range of hosts and locations. We found two genetically distinct variants of PBMYV. PBMYV-1 was the originally described variant, and PBMYV-2 had a large putative recombination in open reading frame 5 such that PBMYV-1 and PBMYV-2 shared only 65-66% amino acid sequence identity in the P5 protein. The virus was transmitted by a clonal colony of cowpea aphids (Aphis craccivora) and by grafting with infected scions but was not transmitted by a clonal colony of green peach aphids (Myzus persicae). PBMYV was found in natural infections in 11 host species with a range of symptoms and severity, including seven important grain legume crops from across a wide geographic area in Australia. PBMYV was common and widespread in the tropical weed phasey bean (Macroptilium lathyroides), but it is likely that there are other major alternative hosts for the virus in temperate regions of Australia. The experimental host range of PBMYV included the Fabaceae hosts chickpea (Cicer arietinum), faba bean (Vicia faba), pea (Pisum sativum), and phasey bean, but transmissions failed to infect several other members of the families Asteraceae, Cucurbitaceae, Fabaceae and Solanaceae. PBMYV was commonly found in grain legume crops in eastern and western Australia, sometimes at greater than 90% incidence. This new knowledge about PBMYV warrants further assessments of its economic impact on important grain legume crops.

Two strains of a novel begomovirus encoding Rep proteins with identical ß1 strands but different ß5 strands are not compatible in replication.

Geminiviruses have genomes composed of single-stranded DNA molecules and encode a rolling-circle replication (RCR) initiation protein ("Rep"), which has multiple functions. Rep binds to specific repeated DNA motifs ("iterons"), which are major determinants of virus-specific replication. The particular amino acid (aa) residues that determine the preference of a geminivirus Rep for specific iterons (i.e., the trans-acting replication "specificity determinants", or SPDs) are largely unknown, but diverse lines of evidence indicate that most of them are closely associated with the so-called RCR motif I (FLTYP), located in the first 12-19 aa residues of the protein. In this work, we characterized two strains of a novel begomovirus, rhynchosia golden mosaic Sinaloa virus (RhGMSV), that were incompatible in replication in pseudorecombination experiments. Systematic comparisons of the Rep proteins of both RhGMSV strains in the DNA-binding domain allowed the aa residues at positions 71 and 74 to be identified as the residues most likely to be responsible for differences in replication specificity. Residue 71 is part of the ß-5 strand structural element, which was predicted in previous studies to contain Rep SPDs. Since the Rep proteins encoded by both RhGMSV strains are identical in their first 24 aa residues, where other studies have mapped potential SPDs, this is the first study lending direct support to the notion that geminivirus Rep proteins contain separate SPDs in their N-terminal domain.

Genome characterization and diversity of trifolium virus 1: identification of a novel legume-infecting capulavirus.

A novel geminivirus was identified in France and Spain in asymptomatic plants of white clover (Trifolium repens) and shrub medick (Medicago arborea). Its genome has the hallmarks of a capulavirus, and its relationship to other capulaviruses was confirmed by phylogenetic analysis. White clover isolates formed a tight cluster in the phylogenetic tree, while shrub medick isolates formed two distinct, more divergent groups with sequence identity values close to the species cutoff. These three groups have likely participated in recombination events involving alfalfa leaf curl virus and French bean severe leaf curl virus. The name "trifolium virus 1" (TrV1) is proposed for this new Capulavirus. Three TrV1 genotypes (TrV1-A, TrV1-B, and TrV1-C) were clearly distinguished.

Aphid transmission of nanoviruses.

The genus Nanovirus consists of plant viruses that predominantly infect legumes leading to devastating crop losses. Nanoviruses are transmitted by various aphid species. The transmission occurs in a circulative nonpropagative manner. It was long suspected that a virus-encoded helper factor would be needed for successful transmission by aphids. Recently, a helper factor was identified as the nanovirus-encoded nuclear shuttle protein (NSP). The mode of action of NSP is currently unknown in contrast to helper factors from other plant viruses that, for example, facilitate binding of virus particles to receptors within the aphids' stylets. In this review, we are summarizing the current knowledge about nanovirus-aphid vector interactions.

Molecular characterization and analysis of conserved potyviral motifs in bean common mosaic virus (BCMV) for RNAi-mediated protection.

Australian bean common mosaic virus (BCMV) isolates were sequenced, and the sequences were compared to global BCMV and bean common mosaic necrosis virus (BCMNV) sequences and analysed for conserved potyviral motifs to generate in planta RNA-interference (RNAi) resistance. Thirty-nine out of 40 previously reported potyvirus motifs were conserved among all 77 BCMV/BCMNV sequences. Two RNAi target regions were selected for dsRNA construct design, covering 920 bp of the nuclease inclusion b (NIb) protein and 461 bp of the coat protein (CP). In silico prediction of the effectiveness of these constructs for broad-spectrum defence against the 77 BCMV and BCMNV sequences was done via analysis of putative 21-nucleotide (nt) and 22-nt small-interfering RNAs (siRNAs) generated from the target regions. The effectiveness of both constructs for siRNA generation and BCMV RNAi-mediated resistance was validated in Nicotiana benthamiana transient assays.

Cucumber mosaic virus Isolates from Greek Legumes are Associated with Satellite RNAs that are Necrogenic for Tomato.

Worldwide, Cucumber mosaic virus (CMV) is the causal agent of many economically important diseases. Based on immunological or molecular analysis, three distinct subgroups of CMV isolates can be identified (IA, IB, and II). In addition, some CMV isolates are associated with satellite RNAs (satRNAs), a type of noncoding transcript that may alter the symptoms of CMV infections. This study presents an analysis of CMV isolates occurring in legumes in Greece in respect to their genetic diversity, and the presence and diversity of their satRNA. Phylogenetic analysis of the CMV coat protein sequence of 18 legume and 5 tomato CMV isolates collected throughout Greece classified them within subgroups IA and IB, with a limited genetic diversity. The CMV satRNAs found in nine field legumes exhibiting mild symptoms and in one tomato with a necrotic syndrome contained a functional necrogenic motif; therefore, they were grouped within the necrogenic group of CMV-satRNAs. The necrotic phenotype was expressed in all legume CMV isolates containing necrogenic satRNAs when mechanically inoculated onto tomato plants. To our knowledge, this is the first observation that legumes host necrogenic CMV-satRNAs. The possible role of legumes in the epidemiology of CMV and necrogenic satRNA complex is discussed.

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.

Desmodium mottle virus, the first legumovirus (genus Begomovirus) from East Africa.

A novel bipartite legumovirus (genus Begomovirus, family Geminiviridae), that naturally infects the wild leguminous plant Desmodium sp. in Uganda, was molecularly characterized and named Desmodium mottle virus. The highest nucleotide identities for DNA-A, obtained from two field-collected samples, were 79.9% and 80.1% with the legumovirus, soybean mild mottle virus. DNA-B had the highest nucleotide identities (65.4% and 66.4%) with a typical non-legumovirus Old World begomovirus, African cassava mosaic virus. This is the first report of a legumovirus in East Africa and extends the known diversity of begomoviruses found infecting wild plants in this continent.

Molecular and biological properties of an endornavirus infecting winged bean (Psophocarpus tetragonolobus).

A double-stranded RNA (dsRNA) of approximately 15 kbp was isolated from asymptomatic winged bean (Psophocarpus tetragonolobus) plants. The size of the dsRNA, together with results of RT-PCR testing, suggested that it was the replicative form of a plant endornavirus. Cloning, sequencing, and sequence analyses confirmed the endornavirus nature of the dsRNA. Conserved motifs typical for endornaviruses were identified and their amino acid sequences compared with those of selected endornaviruses. Phylogenetic analyses revealed a close relationship with Bell pepper endornavirus, Phaseolus vulgaris endornavirus 2, and Hot pepper endornavirus. The dsRNA was present in most P. tetragonolobus genotypes tested. The virus was provisionally named Winged bean endornavirus 1 (WBEV-1).

Construction of an agroinfectious clone of bean rugose mosaic virus using Gibson Assembly.

Construction of agroinfectious viral clones usually requires many steps of cloning and sub-cloning and also a binary vector, which makes the process laborious, time-consuming, and frequently susceptible to some degree of plasmid instability. Nowadays, novel methods have been applied to the assembly of infectious viral clones, and here we have applied isothermal, single-step Gibson Assembly (GA) to construct an agroinfectious clone of Bean rugose mosaic virus (BRMV) using a small binary vector. The procedure has drastically reduced the cloning steps, and BRMV could be recovered from agroinfiltrated common bean twenty days after inoculation, indicating that the infectious clone could spread in the plant tissues and efficiently generate a systemic infection. The virus was also recovered from leaves of common bean and soybean cultivars mechanically inoculated with infectious clone two weeks after inoculation, confirming the efficiency of GA cloning procedure to produce the first BRMV agroinfectious clone to bean and soybean.

Resistance to Bean common mosaic necrosis virus Conferred by the bc-1 Gene Affects Systemic Spread of the Virus in Common Bean.

Bean common mosaic necrosis virus (BCMNV) isolates belong to two pathogroups (PG), PG-III and PG-VI, which are distinguished in common bean due to the inability of the PG-III isolates of BCMNV to overcome the two recessive resistance alleles bc-1 and bc-12. The biological and molecular basis of this distinction between PG-III and PG-VI isolates of BCMNV is not known. Here, three isolates of BCMNV were typed biologically on a set of 12 bean differentials and molecularly through whole-genome sequencing. Two isolates (1755b and TN1a) were assigned to PG-VI and one isolate (NL8-CA) was assigned to PG-III. Isolate NL8-CA (PG-III) induced only local necrosis on inoculated leaves in 'Top Crop' and 'Jubila' bean harboring the I gene and the bc-1 allele, whereas isolates TN1, TN1a, and 1755b (all PG-VI) induced rapid whole-plant necrosis (WPN) in Top Crop 7 to 14 days postinoculation, and severe systemic necrosis but not WPN in Jubila 3 to 5 weeks postinoculation. In 'Redland Greenleaf C' expressing bc-1 and 'Redland Greenleaf B' expressing bc-12 alleles, isolate NL8-CA was able to systemically infect only a small proportion of upper uninoculated leaves (less than 13 and 3%, respectively). The whole genomes of isolates 1755b, TN1a, and NL8-CA were sequenced and sequence analysis revealed that, despite the overall high nucleotide sequence identity between PG-III and PG-VI isolates (approximately 96%), two areas of the BCMNV genome in the P1/HC-Pro and HC-Pro/P3 cistrons appeared to be more divergent between these two pathotypes of BCMNV. The data suggest that the phenotypic differences among PG-III and PG-VI isolates of BCMNV in common bean cultivars from host resistance groups 2, 3, and 9 carrying bc-1 alleles were related to the impaired systemic movement of the PG-III isolates to the upper, uninoculated leaves, and also suggest a role of the recessive bc-1 gene in interfering with systemic spread of BCMNV.

Proteomic analysis of the plasma membrane-movement tubule complex of cowpea mosaic virus.

Cowpea mosaic virus forms tubules constructed from the movement protein (MP) in plasmodesmata (PD) to achieve cell-to-cell movement of its virions. Similar tubules, delineated by the plasma membrane (PM), are formed protruding from the surface of infected protoplasts. These PM-tubule complexes were isolated from protoplasts by immunoprecipitation and analysed for their protein content by tandem mass spectrometry to identify host proteins with affinity for the movement tubule. Seven host proteins were abundantly present in the PM-tubule complex, including molecular chaperonins and an AAA protein. Members of both protein families have been implicated in establishment of systemic infection. The potential role of these proteins in tubule-guided cell-cell transport is discussed.

A novel member of the Tombusviridae from a wild legume, Gompholobium preissii.

As part of an investigation into viruses of wild plants in Australia, a contiguous sequence of 3935 nucleotides was obtained after shotgun sequencing of RNA isolated from an asymptomatic wild legume, Gompholobium preissii. Phylogenetic analysis of the sequence revealed that it most closely resembled that of Trailing lespedeza virus 1 (TLV1), a virus isolated from a wild legume in America. The proposed virus, named Gompholobium virus A, and TLV1 are genetically closest to viruses in the genera Alphacarmovirus and Pelarspovirus, family Tombusviridae, but they share features distinguishing them from both groups.

Seed-borne nature of a begomovirus, Mung bean yellow mosaic virus in black gram.

The yellow mosaic viruses (YMV) infecting legumes are considered to be the most devastating begomoviruses as they incite considerable yield loss. The yellow discoloration of pods and seeds of infected plants and symptom emergence in the very first trifoliate leaf of the plants in the field were suggestive that the virus may be seed borne, which was investigated in the present study. The distribution of the virus in various parts of the seeds of black gram (Vigna mungo L. Hepper) plants naturally infected in the field was determined by polymerase chain reaction (PCR), Southern blot analysis, and sequencing. Nucleotide sequencing of the PCR amplicons from the seed parts from groups of ten seeds revealed the presence of mung bean yellow mosaic virus (MYMV) in the seed coat, cotyledon, and embryonic axes. The presence of virion particles was confirmed through double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and immunosorbent electron microscopy (ISEM) even in a single whole seed. In confocal microscopy, positive fluorescent signals were obtained using coat protein gene-specific primers in the embryonic axes. However, in the growth tests performed with the same batch of seeds, there was no symptom development in the seedlings though the virus (both DNA A and B components) was detected in 32 % of tested seedlings. In this study, the MYMV was detected in seed coat, cotyledon, and embryo. This study revealed that the MYMV is a seed-borne virus.

Camelid nanobodies with high affinity for broad bean mottle virus: a possible promising tool to immunomodulate plant resistance against viruses.

Worldwide, plant viral infections decrease seriously the crop production yield, boosting the demand to develop new strategies to control viral diseases. One of these strategies to prevent viral infections, based on the immunomodulation faces many problems related to the ectopic expression of specific antibodies in planta. Camelid nanobodies, expressed in plants, may offer a solution as they are an attractive tool to bind efficiently to viral epitopes, cryptic or not accessible to conventional antibodies. Here, we report a novel, generic approach that might lead to virus resistance based on the expression of camelid specific nanobodies against Broad bean mottle virus (BBMV). Eight nanobodies, recognizing BBMV with high specificity and affinity, were retrieved after phage display from a large 'immune' library constructed from an immunized Arabic camel. By an in vitro assay we demonstrate how three nanobodies attenuate the BBMV spreading in inoculated Vicia faba plants. Furthermore, the in planta transient expression of these three selected nanobodies confirms their virus neutralizing capacity. In conclusion, this report supports that plant resistance against viral infections can be achieved by the in vivo expression of camelid nanobodies.

Role of electrostatics in the assembly pathway of a single-stranded RNA virus.

UNLABELLED: We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318-3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of all of the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively charged arginine-rich motifs (ARMS) of the CPs with the negatively charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a charge-matched protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally symmetric nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario, we prepare CP mutants with extra and missing (relative to the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA. IMPORTANCE: Cowpea chlorotic mottle virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly, i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA driven by electrostatic attraction, followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of such a mechanism by demonstrating that efficient assembly is initiated by the formation of a disordered protocapsid complex whose stoichiometry is governed by electrostatics (charge matching of the anionic RNA and the cationic N termini of the CP).

The AC5 protein encoded by Mungbean yellow mosaic India virus is a pathogenicity determinant that suppresses RNA silencing-based antiviral defenses.

It is generally accepted that begomoviruses in the family Geminiviridae encode four proteins (from AC1/C1 to AC4/C4) using the complementary-sense DNA as template. Although AC5/C5 coding sequences are increasingly annotated in databases for many begomoviruses, the evolutionary relationships and functions of this putative protein in viral infection are obscure. Here, we demonstrate several important functions of the AC5 protein of a bipartite begomovirus, Mungbean yellow mosaic India virus (MYMIV). Mutational analyses and transgenic expression showed that AC5 plays a critical role in MYMIV infection. Ectopic expression of AC5 from a Potato virus X (PVX) vector resulted in severe mosaic symptoms followed by a hypersensitive-like response in Nicotiana benthamiana. Furthermore, MYMIV AC5 effectively suppressed post-transcriptional gene silencing induced by single-stranded but not double-stranded RNA. AC5 was also able to reverse transcriptional gene silencing of a green fluorescent protein transgene by reducing methylation of promoter sequences, probably through repressing expression of a CHH cytosine methyltransferase (DOMAINS REARRANGED METHYLTRANSFERASE2) in N. benthamiana. Our results demonstrate that MYMIV AC5 is a pathogenicity determinant and a potent RNA silencing suppressor that employs novel mechanisms to suppress antiviral defenses, and suggest that the AC5 function may be conserved among many begomoviruses.

Engineered single-chain variable fragment antibody for immunodiagnosis of groundnut bud necrosis virus infection.

Few studies have been done on engineered antibodies for diagnosis of tospovirus infections. The present study was undertaken to develop a single-chain variable fragment (scFv) for specific diagnosis of infection by groundnut bud necrosis virus (GBNV), the most prevalent serogroup IV tospovirus in India. Heavy chain (372 nucleotide [nt]) and light chain (363 nt) variable region clones obtained from a hybridoma were used to make an scFv construct that expressed a ~29-kDa protein in E. coli. The scFv specifically detected GBNV in field samples of cowpea, groundnut, mung bean, and tomato, and it did not recognize watermelon bud necrosis virus, a close relative of GBNV belonging to tospovirus serogroup IV. This study for the first time demonstrated the application of a functional scFv against a serogroup-IV tospovirus.