Cnidoscolus mild mosaic virus: a new bipartite begomovirus isolated from Cnidoscolus urens in Brazil.

A novel bipartite begomovirus infecting Cnidoscolus urens (Euphorbiaceae) from Pernambuco State, Brazil, has been characterized. The complete DNA-A (2657 nt) and DNA-B (2622 nt) components of the viral isolates show the typical genome organization of New World bipartite begomoviruses. DNA-A of the isolates had the highest percentage of nucleotide sequence identity (88.6-88.9%) to cnidoscolus mosaic leaf deformation virus. Based on the current classification criteria for the genus Begomovirus, the virus infecting C. urens should be considered a new member of the genus, and the name "cnidoscolus mild mosaic virus" is proposed for the virus, and the name "Begomovirus caboniensis" is proposed for its species.

Identification of Cacao Mild Mosaic Virus (CaMMV) and Cacao Yellow Vein-Banding Virus (CYVBV) in Cocoa (Theobroma cacao) Germplasm.

Cocoa, Theobroma cacao, is an important tropical perennial crop grown widely in the humid tropics. The exchange of cocoa germplasm between germplasm collections and breeding centres is vital for varietal development. Intermediate quarantine facilities, such as the International Cocoa Quarantine Centre, Reading UK (ICQC-R) play a vital role in ensuring the transfer of germplasm whilst minimising the risk of spreading pests and diseases. Current screening procedures combine visual inspection and molecular techniques, which are effective in detecting Cocoa swollen shoot virus (CSSV), a badnavirus, which causes severe losses but are restricted to West Africa. However, the detection of latent or mild virus infections that produce no visual symptoms has been a challenge. Recently two badnavirus species of cocoa producing mild symptoms, cacao mild mosaic virus (CaMMV) and cacao yellow vein-banding virus (CYVBV), have been sequenced. Here, we report new assays for the detection of these two species, for the first time in non-symptomatic accessions. Evolutionary and bioinformatic analyses of the viruses suggest their most recent source was from Trinidad, though there is historic evidence that these viruses may have their origin in South America and then become widespread globally over the last century. We also report a novel colorimetric Loop-mediated isothermal amplification (LAMP) assay for the detection of CYVBV. This simple and accurate method could be employed in field virus testing.

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins.

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.

Complete sequence of an isolate of snake melon asteroid mosaic virus confirms that it is a member of a distinct sobemovirus species.

A virus tentatively named "snake melon asteroid mosaic virus" (SMAMV) was found in Sudan in cucurbit crops (10% of 600 samples) between 1992 and 2003. Biological and cytological properties as well as sequence data on a 345-nt fragment suggested that SMAMV was a member of the genus Sobemovirus. However, no complete sequence had been obtained, and the relationship between SMAMV and the acknowledged sobemoviruses had not been ascertained. In this work, we obtained the full-length sequence of an SMAMV isolate. The sequence was 4225 nt long, with a typical sobemovirus genetic organization. Sequence identity to other sobemoviruses was below 50%, both for the full-length genome and for individual proteins. These data confirm that SMAMV belongs to a novel sobemovirus species.

Complete nucleotide sequence of chrysanthemum mosaic-associated virus, a novel emaravirus infecting chrysanthemum.

Here, we report the complete genome sequence of chrysanthemum mosaic-associated virus (ChMaV), a putative new member of the genus Emaravirus. The ChMaV genome comprises seven negative-sense RNA segments (RNAs 1, 2, 3a, 3b, 4, 5, and 6), and the amino acid sequences of its RNA-dependent RNA polymerase (RNA1), glycoprotein precursor (RNA2), nucleocapsid protein (RNA3), and movement protein (RNA4) showed the closest relationship to pear chlorotic leaf spot-associated virus. Phylogenetic analysis showed that it clusters with emaraviruses whose host plants originate from East Asia.

Costus stripe mosaic virus, a tentative new member of the genus Potyvirus.

Morphological, biological, serological, and molecular tests underpin the description of costus stripe mosaic virus (CoSMV) as a new member of the genus Potyvirus, family Potyviridae. Found affecting the native ornamental Costus spiralis in Brazil, the pathogen showed a severely restricted natural and experimental host range. Excluding the poly(A) tail, the CoSMV genome contains a large open reading frame (ORF) of 9,446 nucleotides that encodes a polyprotein with 3,046 amino acids, which is potentially cleaved into ten products, and a small ORF (77 amino acids) knows as PIPO. Genome analysis demonstrated the highest CoSMV nucleotide sequence identity to onion yellow dwarf virus (51.79%). No evidence of recombination was detected in the CoSMV genome, and phylogenetic analysis revealed its basal position in a group formed by members of the genus Potyvirus, along with Cyrtanthus elatus virus A (Vallota speciosa virus) and canna yellow streak virus. CoSMV was not transmitted by aphids of the species Aphis solanella, Myzus persicae or Uroleucon sonchi, which could be due to mutations in the HC-Pro motifs required for aphid transmission. A divergence in the P1 protein cleavage site was found when compared to other members of the family Potyviridae. Based on its unique biological and molecular characteristics and the current species demarcation criteria, we propose CoSMV to be a new tentative member of the genus Potyvirus.

Molecular and biological characterization of a putative new sobemovirus infecting Physalis peruviana.

Physalis peruviana is a perennial solanaceous plant that has recently been established as a commercial crop in Brazil. This work reports the near-complete genome sequence, particle morphology, and plant host responses to a putative new sobemovirus, named "physalis rugose mosaic virus". The virus, characterized by isometric particles of ca. 30 nm in diameter, causes foliar symptoms of mosaic, malformation and blistering, accompanied by stunting. The near-complete genome sequence comprises 4175 nucleotides and contains five open reading frames that are similar to those of other sobemoviruses. In addition to P. peruviana, the new virus systemically infected Capsicum annuum, Nicotiana tabacum and Solanum lycopersicum by mechanical inoculation. Thus, this virus may cause disease in these crops in the field.

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.

Strain-dependent mutational effects for Pepino mosaic virus in a natural host.

BACKGROUND: Pepino mosaic virus (PepMV) is an emerging plant pathogen that infects tomatoes worldwide. Understanding the factors that influence its evolutionary success is essential for developing new control strategies that may be more robust against the evolution of new viral strains. One of these evolutionary factors is the distribution of mutational fitness effect (DMFE), that is, the fraction of mutations that are lethal, deleterious, neutral, and beneficial on a given viral strain and host species. The goal of this study was to characterize the DMFE of introduced nonsynonymous mutations on a mild isolate of PepMV from the Chilean 2 strain (PepMV-P22). Additionally, we also explored whether the fitness effect of a given mutation depends on the gene where it appears or on epistatic interactions with the genetic background. To address this latter possibility, a subset of mutations were also introduced in a mild isolate of the European strain (PepMV-P11) and the fitness of the resulting clones measured. RESULTS: A collection of 25 PepMV clones each containing a single nucleotide nonsynonymous substitution was created by site-directed mutagenesis and the fitness of each mutant was determined. PepMV-P22 genome showed a high degree of robustness against point mutations, with 80% of mutations being either neutral or even beneficial and only 20% being deleterious or lethal. We found that the effect of mutations strongly depended on the gene in which they were introduced. Mutations with the largest average beneficial effects were those affecting the RdRp gene, in contrast to mutations affecting TGB1 and CP genes, for which the average effects were deleterious. Moreover, significant epistatic interactions were observed between nonsynonymous mutations and the genetic background, meaning that the effect of a given nucleotide substitution on a particular genomic context cannot be predicted by knowing its effect in a different one. CONCLUSIONS: Our results indicated that PepMV genome has a surprisingly high robustness against mutations. We also found that fitness consequences of a given mutation differ between the two strains analyzed. This discovery suggests that the strength of selection, and thus the rates of evolution, vary among PepMV strains.

Complete genome sequences of two highly divergent Japanese isolates of Plantago asiatica mosaic virus.

Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus and has an exceptionally wide host range. It causes severe damage to lilies. Here we report on the complete nucleotide sequences of two new Japanese PlAMV isolates, one from the eudicot weed Viola grypoceras (PlAMV-Vi), and the other from the eudicot shrub Nandina domestica Thunb. (PlAMV-NJ). Their genomes contain five open reading frames (ORFs), which is characteristic of potexviruses. Surprisingly, the isolates showed only 76.0-78.0 % sequence identity with each other and with other PlAMV isolates, including isolates from Japanese lily and American nandina. Amino acid alignments of the replicase coding region encoded by ORF1 showed that the regions between the methyltransferase and helicase domains were less conserved than other regions, with several insertions and/or deletions. Phylogenetic analyses of the full-length nucleotide sequences revealed a moderate correlation between phylogenetic clustering and the original host plants of the PlAMV isolates. This study revealed the presence of two highly divergent PlAMV isolates in Japan.

Characterization of a new carmovirus isolated from an Adonis plant.

An isometric virus was isolated from a cultivated Adonis plant (A. ramosa). The purified virus particle is 28 nm in diameter and is composed of a single coat protein and a single RNA genome of 3,991 nucleotides. Sequence analysis showed that the virus is closely related to carnation mottle virus. The virus was used to mechanically infect healthy A. ramosa plants, resulting in mosaic and leaf curl symptoms; however, attempts to inoculate carnation plants did not result in infection. We propose the virus as a new carmovirus and have named it adonis mosaic virus (AdMV).

Biological and molecular characterization of a putative new potexvirus infecting Senna occidentalis.

In this work, we report the complete genome sequence of, production of polyclonal antibodies against, and development of biological assays for a putative new potexvirus, named senna mosaic virus (SenMV), found infecting Senna occidentalis in the state of São Paulo, Brazil. The complete genome sequence of SenMV comprises 6775 nucleotides excluding the poly(A) tail. The genome organization is similar to those of other potexviruses, with five open reading frames coding for RNA-dependent RNA polymerase (RdRp), the triple gene block (TGB 1, 2, and 3) proteins, and coat protein (CP). The virus was transmitted to S. occidentalis by mechanical inoculation and trimming scissors, but not by seeds.

A dCAPS assay detects and characterizes BaYMV according to its ability to overcome rym4-mediated resistance.

Winter barley is subjected to severe yield losses due to the yellow mosaic virus disease. Two soil borne bymoviruses, BaYMV (Barley yellow mosaic virus) and BaMMV (Barley mild mosaic virus) are responsible for this disease in Europe. As breeding resistant cultivars is the only control method against the disease, barley varieties carrying the recessive resistance rym4 were introduced. However, a new pathotype BaYMV-2 overcoming rym4 resistance appeared in the late 1980s. In France, little is known about BaYMV-2 and the common BaYMV (BaYMV-1) distribution, but the increase of the disease occurrence is becoming a concern. There is currently no valid molecular tool for BaYMV-1 and BaYMV-2 differentiation; thus the development of a dCAPS (derived Cleaved Amplified Polymorphic Sequences) tool was investigated. BaYMV-1 and BaYMV-2 diversity was first estimated by Sanger sequencing. The Single Nucleotide Polymorphism (SNP) previously described as providing the ability to overcome rym4-mediated resistance was targeted. A dCAPS tool was developed to digest specifically BaYMV-1. This assay was successfully tested with seventy naturally infected samples. This new tool will be useful to investigate BaYMV-1 and 2 distributions.

Molecular Variability and Distribution of Sugarcane Mosaic Virus in Shanxi, China.

BACKGROUND: Sugarcane mosaic virus (SCMV) is responsible for large-scale economic losses in the global production of sugarcane, maize, sorghum, and some other graminaceous species. To understand the evolutionary mechanism of SCMV populations, this virus was studied in Shanxi, China. A total of 86 maize leaf samples (41 samples in 2012 and 45 samples in 2013) were collected from 4 regions of Shanxi. RESULTS: Double-antibody sandwich (DAS)-ELISA and RT-PCR showed 59 samples (30 samples in 2012 and 29 samples in 2013) to be positive for SCMV, from which 10 new isolates of SCMV were isolated and sequenced. The complete genomes of these isolates are 9610 nt long, including the 5' and 3' non-coding regions, and encode a 3063-amino acid polyprotein. Phylogenetic analyses revealed that 24 SCMV isolates could be divided on the basis of the whole genome into 2 divergent evolutionary groups, which were associated with the host species. Among the populations, 15 potential recombination events were identified. The selection pressure on the genes of these SCMV isolates was also calculated. The results confirmed that all the genes were under negative selection. CONCLUSIONS: Negative selection and recombination appear to be important evolutionary factors shaping the genetic structure of these SCMV isolates. SCMV is distributed widely in China and exists as numerous strains with distinct genetic diversity. Our findings will provide a foundation for evaluating the epidemiological characteristics of SCMV in China and will be useful in designing long-term, sustainable management strategies for SCMV.

Sequence diversity of wheat mosaic virus isolates.

Wheat mosaic virus (WMoV), transmitted by eriophyid wheat curl mites (Aceria tosichella) is the causal agent of High Plains disease in wheat and maize. WMoV and other members of the genus Emaravirus evaded thorough molecular characterization for many years due to the experimental challenges of mite transmission and manipulating multisegmented negative sense RNA genomes. Recently, the complete genome sequence of a Nebraska isolate of WMoV revealed eight segments, plus a variant sequence of the nucleocapsid protein-encoding segment. Here, near-complete and partial consensus sequences of five more WMoV isolates are reported and compared to the Nebraska isolate: an Ohio maize isolate (GG1), a Kansas barley isolate (KS7), and three Ohio wheat isolates (H1, K1, W1). Results show two distinct groups of WMoV isolates: Ohio wheat isolate RNA segments had 84% or lower nucleotide sequence identity to the NE isolate, whereas GG1 and KS7 had 98% or higher nucleotide sequence identity to the NE isolate. Knowledge of the sequence variability of WMoV isolates is a step toward understanding virus biology, and potentially explaining observed biological variation.

Analysis of genome comparison of two Indian isolates of Cowpea aphid-borne mosaic virus from India.

The complete sequence of two Cowpea aphid-borne mosaic virus (CABMV) isolates (RR3 and RR4) from India was determined. Phylogenetic analysis showed that both isolates showed different closeness with other isolates of CABMV. CABMV-RR3 showed maximum identity of 99 % with CABMV-BR1 from Brazil at nucleotide and protein levels, whereas CABMV-RR4 showed identity of 73 and 95 % with CABMV-Z isolate from Zimbabwe at nucleotide and protein levels respectively. Similarity identity matrix revealed 69 % identity at nucleotide level and 91 % at protein level with each other. Recombination breakpoint detection showed that CABMV-MG-Avr from Brazil and CABMV-Z from Zimbabwe act as major parents in our isolates RR3 and RR4, respectively.

Pigeonpea sterility mosaic virus: a legume-infecting Emaravirus from South Asia.

Pigeonpea sterility mosaic virus (PPSMV), a species of the genus Emaravirus, is the causal agent of sterility mosaic disease (SMD) of pigeonpea [Cajanus cajan (L.) Millsp]. This disease, dubbed the 'green plague', as the infected plants remain in the vegetative state without flower production, has been reported from India and a few other South-East Asian countries. SMD is estimated to result in an annual yield loss of over US$300 million in India alone. The aetiology of SMD, which remained a mystery for over 70 years, was resolved with the discovery of PPSMV in 2000 and its complete genome sequence in 2014. AETIOLOGY AND VIRUS TRANSMISSION: SMD is characterized by stunted and bushy plants, leaves of reduced size with chlorotic rings or mosaic symptoms, and partial or complete cessation of flower production (i.e. sterility). The causal agent of the disease is PPSMV, a virus with a segmented, negative-sense, single-stranded RNA genome, transmitted in a semi-persistent manner by an eriophyid mite Aceria cajani Channabassavanna (Acari: Arthropoda). Both the virus and vector are highly specific to pigeonpea and a few of its wild relatives, such as C. scarabaeoides and C. cajanifolius. Under experimental conditions, PPSMV was transmitted to Nicotiana benthamiana by sap inoculation using fresh extract of SMD-infected leaves (but not to pigeonpea); however, purified nucleoprotein preparations are not infectious. The virus was also transmitted to French bean (Phaseolus vulgaris L.) using viruliferous eriophyid mites. PPSMV is not seed transmitted in pigeonpea or other hosts known to be infected by this virus. On the basis of the differential host reactions in different geographical locations, the occurrence of diverse PPSMV strains was suspected. HOST RANGE AND EPIDEMIOLOGY: PPSMV can infect several genotypes of cultivated and wild relatives of pigeonpea. Experimental hosts include N. benthamiana, N. clevelandii, P. vulgaris and Chrozophora rottleri. However, pigeonpea alone and a few wild species of Cajanus were found to support the vector A. cajani. SMD is endemic in most of the pigeonpea-growing regions of India, but the incidence varies widely between regions and years. In nature, A. cajani populations were almost exclusively observed on SMD-infected pigeonpea, but not on healthy plants, indicating a strong communalistic relationship between the virus-infected plants and the vector. The epidemiology of SMD involves the virus, mite vector, cultivar and environmental conditions. Infected perennial and volunteer plants serve as a source for both the virus and its vector mites, and play an important role in the disease cycle. GENOME ORGANIZATION, GENE FUNCTION AND TAXONOMY: The PPSMV genome contains five segments of single-stranded RNA that are predicted to encode proteins in negative sense. The ribonucleoprotein complex is encased in quasi-spherical, membrane-bound virus particles of 100-150 nm. The largest segment, RNA-1, is 7022 nucleotides in length and codes for RNA-dependent RNA polymerase (2295 amino acids); RNA-2, with a sequence length of 2223 nucleotides, codes for glycoproteins (649 amino acids); RNA-3, with a sequence length of 1442 nucleotides, codes for nucleocapsid protein (309 amino acids); RNA-4, with a sequence length of 1563 nucleotides, codes for a putative movement protein p4 (362 amino acids); and RNA-5, with a sequence length of 1689 nucleotides, codes for p5 (474 amino acids), a protein with unknown function. PPSMV was recently classified as a species in the genus Emaravirus, a genus whose members show features resembling those of members of the genera Tospovirus (Family: Bunyaviridae) and Tenuivirus, both of which comprise single-stranded RNA viruses that encode proteins by an ambisense strategy. SMD CONTROL: The disease is mainly controlled using SMD-resistant cultivars. However, the occurrence of distinct strains/isolates of PPSMV in different locations makes it difficult to incorporate broad-spectrum resistance. Studies on the inheritance of SMD resistance in different cultivars against different isolates of PPSMV indicate that the resistance is mostly governed by recessive genes, although there are contrasting interpretations of the data. Genetic engineering through RNA-interference (RNAi) and resistant gene-based strategies are some of the potential approaches for the transgenic control of SMD. Seed treatment or soil and foliar application of a number of organophosphorus-based insecticides or acaricides, which are recommended for the management of the vector mites, are seldom practised because of prohibitive costs and also their risks to human health and the environment.

Development of a one-step immunocapture real-time RT-PCR assay for the detection of barley stripe mosaic virus strains in barley seedlings.

A one-step immunocapture real-time RT-PCR (IC-real-time RT-PCR) was developed for efficient detection of barley stripe mosaic virus (BSMV) in barley seedlings. The novel detection system was designed using a primer set targeting the conserved region in the triple gene block 2 (TGB2) to expand its capacity to detect all BSMV strains. This assay was evaluated for its efficiency in detecting BSMV in barley seedlings. Using the immunocapture sample preparation, real-time RT-PCR was able to detect BSMV in samples, which were indicated as negative by ELISA. The sensitivity of detection in the real-time RT-PCR was as low as 50 fg/µl of total viral RNA under optimal reaction conditions. This level of sensitivity indicated that the one-step IC-real-time RT-PCR developed in the present study could be used for routine plant and seed health assays.

Complete genome sequence of Jacquemontia yellow mosaic virus, a novel begomovirus from Venezuela related to other New World bipartite begomoviruses infecting Convolvulaceae.

The complete genome of a bipartite begomovirus (genus Begomovirus, family Geminiviridae) infecting Jacquemontia sp. (Convolvulaceae) in Venezuela has been cloned and sequenced. Sequence comparison and phylogenetic analysis have shown that it represents an isolate of a novel species with closest relatives being two New World bipartite begomoviruses that infect Convolvulaceae, Jacquemontia mosaic Yucatan virus and Merremia mosaic virus. The DNA-As of these begomoviruses, however, share only 77.0-78.4 % nucleotide sequence identity with the DNA-A of the isolate described here, for which a recombinant origin is suggested. Based on the symptoms observed in the field, the name Jacquemontia yellow mosaic virus (JacYMV) is proposed for this novel bipartite begomovirus.

Sequence-modified primers for the differential RT-PCR detection of Andean potato latent and Andean potato mild mosaic viruses in quarantine tests.

To enable the differential PCR detection of Andean potato latent virus (APLV) and Andean potato mild mosaic virus (APMMV) strains, sense primers were designed that correspond to regions directly upstream of the coat protein genes. Their differentiating power was increased by A->C or T->C replacements in their 3'-terminal parts. Together with the broad-specificity antisense primer EM3, primer AL-a-mod3C detected all APLV strains tested, but none of the APMMV strains. Primer AM-a-mod4C yielded PCR products with all APMMV preparations, but also with some APLV preparations. Sequence analysis revealed that this was not due to a lack of primer specificity, but to the sensitive detection of contaminating APMMV in some of our APLV preparations.