Dwarf Virus Isolates Research Articles

Genetic Variation and Evolutionary Analysis of Eggplant Mottled Dwarf Virus Isolates from Spain.

The genetic variation and population structure of gene N (nucleocapsid) and part of gene L (replicase) from 13 eggplant mottle dwarf virus (EMDV) isolates from Spain were evaluated and compared with sequences of EMDV isolates from other countries retrieved from GenBank. Phylogenetic inference of part of gene L showed three main clades, one containing an EMDV isolate from Australia and the other two containing isolates from Iran and Europe, as well as four subclades. EMDV isolates from Spain were genetically very similar and grouped in a subclade together with one isolate from Germany and one from the UK. No new recombination events were detected in addition to one recombination previously reported, suggesting that recombination is rare for EMDV. The comparison of synonymous and non-synonymous rates showed that negative selection played an important role, and only two codons were under positive selection. Genetic differentiation (Fst test), phylogenetic and nucleotide diversity analyses suggest a unique introduction of EMDV to Spain and low gene flow with other countries. In contrast, Greece and Italy showed diverse populations with high gene flow between both.

Global phylogenetic analysis of soybean dwarf virus isolates and their associations with aphid vectors and severe disease in soybeans.

Soybean dwarf virus (SbDV) was first described in Japan as an agent of severe soybean disease transmitted by the foxglove aphid, Aulacorthum solani, with separable yellowing (Y) and dwarfing (D) strains. SbDV of both Y and D genotypes were later documented in other countries. For three decades, SbDV isolates were assessed to evaluate risk to U.S. soybean production. U.S. SbDV isolates were transmitted by the pea aphid Acyrthosiphum pisum and showed limited disease in soybeans, suggesting it was not a major threat to U.S. soybean production. Here we report 21 new full-length SbDV genome sequences including those of the originally described Japanese Y and D isolates, isolates from Syria and New Zealand associated with severe disease, and 17 isolates from U.S. field collections. Using these new full-length genomes, a global phylogeny was assembled and used to revisit risk assessment based on sequence similarities, isolate pathogenicity, and vector specificity.

Molecular characterization of the partial coat protein gene of an Onion yellow dwarf virus isolate detected in garlic (Allium sativum L.) from the West Shewa zone of Ethiopia

Molecular characterization of the partial coat protein gene of an Onion yellow dwarf virus isolate detected in garlic (Allium sativum L.) from the West Shewa zone of Ethiopia

Vector species, pasture legume host range, and impact on grain legumes of an Australian soybean dwarf virus isolate.

Soybean dwarf virus (SbDV; family Tombusviridae, genus Luteovirus, species Soybean dwarf virus) can cause damaging disease epidemics in cultivated plants of the family Fabaceae. The biological characteristics of SbDV isolate WA-8, including its vector species, host range, and impact on Australian grain legume cultivars, were investigated in a series of glasshouse experiments. Isolate WA-8 was classified as the YP strain, as it was transmitted by Acyrthosiphon pisum (pea aphid) and Myzus persicae (green peach aphid) and infected known strain indicator species. Of the 18 pasture legume species inoculated with SbDV, 12 were SbDV hosts, including eight that had not been identified previously as hosts. When inoculated with SbDV, field pea (Pisum sativum), faba bean (Vicia faba), lentil (Lens culinaris), and narrow-leafed lupin cv. Jurien were the most susceptible (70 to 100% plant infection rates), and albus lupin (Lupinus albus), chickpea (Cicer arietinum), and narrow-leafed lupin cv. Mandelup were less susceptible (20 to 70%). Over the course of three experiments, chickpea was the most sensitive to infection, with a > 97% reduction in dry above-ground biomass (AGB) and a 100% reduction in seed yield. Field pea cv. Gunyah, faba bean, and lentil were also sensitive, with a 36 to 61% reduction in AGB. Field pea cv. Kaspa was relatively tolerant, with no significant reduction in AGB or seed yield. The information generated under glasshouse conditions in this study provides important clues for understanding SbDV epidemiology and suggests that it has the potential to cause damage to Australian grain legume crops in the field, especially if climate change facilitates its spread.

Aphid transmissibility of onion yellow dwarf virus isolates with an N-terminal truncated HC-Pro is aided by leek yellow stripe virus

We recently reported that most of the onion yellow dwarf virus (OYDV) isolates detected from garlic in Hokkaido, Japan have a short HC-Pro. Because HC-Pro has been found to be necessary for aphid transmission, OYDV with a short HC-Pro (OYDV-S) conceivably may have lost aphid transmissibility. We here confirmed that OYDV-S was transmitted by aphids only when coinfecting the host with another potyvirus, leek yellow stripe virus (LYSV); OYDV with an intact HC-Pro was transmitted by aphids without LYSV. We presume that LYSV HC-Pro works in trans as a platform that interlinks both LYSV and OYDV-S with the aphid stylet.

Complete Genome Sequence of a Soybean Dwarf Virus Isolate from White Clover in Germany.

In this study, we present the complete genome of a new isolate of soybean dwarf virus (SbDV) (genus Luteovirus, family Luteoviridae) from white clover in Germany. The complete genome of the isolate (JKI ID 23556) consists of 5,858 nucleotides and displays 94.98% nucleotide identity to its most similar SbDV relative (GenBank accession number MN412736).

Complete genome sequences and recombination analysis of three divergent Satsuma dwarf virus isolates

Citrus mosaic virus (CiMV) and navel orange infectious mottling virus (NIMV) are considered strains of satsuma dwarf virus (SDV). To understand the distribution of different isolates of SDV in China, a field survey was conducted, which allowed for the detection of CiMV and NIMV in citrus plants in Zhejiang and Sichuan provinces, respectively. In the present work, two CiMV isolates, including one named CiMV-GTC1 from Goutoucheng sour orange and one named CiMV-SE1 from Setoka tangor, as well as one NIMV isolate named NIMV-EH1 from Ehime-ken No. 38 tangor were, for the first time, fully sequenced. Among the isolates, CiMV-SE1 and CiMV-GTC1 were closely associated with the partial genomes of known CiMV isolates, while NIMV-EH1 was markedly different from known NIMV isolate. Sequence analysis demonstrated that CiMV, NIMV, and SDV shared similar genome organization and high nucleotide identities at the genome level. In addition, phylogenetic reconstruction of the three viruses also suggested a close relationship. Furthermore, recombination analysis provided evidence for their common co-occurrence in nature. These findings contribute significantly to our understanding of the natural evolution of SDV populations and can provide a basis for preventing the spread of different SDV isolates in the main citrus production regions.

Molecular detection and characterisation of black raspberry necrosis virus and raspberry bushy dwarf virus isolates in wild raspberries

AbstractVirus‐derived small interfering RNAs (siRNAs) were extracted from leaves of wild raspberries (Rubus idaeus) sampled from three different regions in Finland and subjected to deep sequencing. Assembly of the siRNA reads to contigs and their comparison to sequences in databases revealed the presence of the bipartite positive‐sense single‐stranded RNA viruses, raspberry bushy dwarf virus (RBDV, genus Idaeovirus), and black raspberry necrosis virus (BRNV, family Secoviridae) in 19 and 26 samples, respectively, including 15 plants coinfected with both viruses. Coverage with siRNA reads [21 and 22 nucleotides (nt)] was higher in BRNV‐FI (Finland) RNA1 (79%) than RNA2 (45%). In RBDV, the coverage of siRNA reads was 89% and 90% for RNA1 and RNA2, respectively. Average depth of coverage was 1.6–4.9 for BRNV and 16.5–36.5 for RBDV. PCR primers designed for RBDV and BRNV based on the contigs were used for screening wild raspberry and a few cultivated raspberry samples from different regions. Furthermore, the sequences of BRNV RNA1 and RNA2 were determined by amplification and sequencing of overlapping contigs (length 1000–1200 nt) except for the 3′ and 5′ ends of RNA1 and RNA2 covered by primers. RNA1 of the Finnish BRNV isolate (BRNV‐FI) was 80% and 86% identical to BRNV‐NA (USA) and BRNV‐Alyth (UK), respectively, whereas the identity of NA and Alyth was 79%. RNA2 of BRNV‐FI was 84% and 80% identical to BRNV‐NA and BRNV‐Alyth, respectively, whereas NA and Alyth were 82% identical. Hence, the strains detected in Finland differ from those reported in the UK and USA. Our results reveal the presence of BRNV in Finland for the first time. The virus is common in wild raspberries and nearly identical isolates are found in cultivated raspberries as well. The results show that wild raspberries in Finland are commonly infected with RBDV or BRNV or both viruses and thus are likely to serve as reservoirs of RBDV and BRNV for cultivated Rubus spp.

Complete genomic characterization of milk vetch dwarf virus isolates from cowpea and broad bean in Anhui province, China.

Cowpea and broad bean plants showing severe stunting and leaf rolling symptoms were observed in Hefei city, Anhui province, China, in 2014. Symptomatic plants from both species were shown to be infected with milk vetch dwarf virus (MDV) by PCR. The complete genomes of MDV isolates from cowpea and broad bean were sequenced. Each of them had eight genomic DNAs that differed between the two isolates by 10.7% in their overall nucleotide sequences. In addition, the MDV genomes from cowpea and broad bean were associated with two and three alphasatellite DNAs, respectively. This is the first report of MDV on cowpea in China and the first complete genome sequences of Chinese MDV isolates.

Phylogenetic analysis of Wheat dwarf virus isolates from Iran.

Wheat dwarf virus (WDV) adversely affects cereal production in Asia, Europe, and North Africa. In this study, sequences of several WDV isolates from Iran which is located in the Fertile Crescent were analyzed. Analysis revealed a new geographic cluster for WDV-Wheat from Iran. Recombination analysis demonstrated the existence of several breakpoints in different regions of the viral genome. Data analysis demonstrated that WDV-Barley has an older history and lower diversity than WDV-Wheat. Sequence analysis identified a rare occasion of a co-infection of wheat with WDV-Wheat and WDV-Barley.

Sequence variation of block III segment identifies three distinct lineages within Eggplant mottled dwarf virus isolates from Italy, Spain and Greece.

Partial polymerase (L) gene sequences of 919 nts, including the conserved segments pre-motif Aand motif Aof block III, of 20 Eggplant mottled dwarf virus (EMDV) isolates were generated, and trimmed sequences of 889 nts, based on the length of available sequences of other isolates, were used to determine phylogenetic relationships. Phylogenetic reconstructions revealed two divergent lineages, designated as genetic group A(Italian isolates) and group B, with the latter further divided into subgroups BI (Greek isolates) and BII (Spanish isolates). No evidence of recombination signals among sequences was detected, whereas analysis of the nonsynonymous/synonymous ratio indicated strong purifying selection, with codons under negative selection uniformly distributed along the sequences. An RT-PCR-RFLP method able to discriminate EMDV isolates of the two main genetic groups was proposed.

Function and diversity of P0 proteins among cotton leafroll dwarf virus isolates.

BackgroundThe RNA silencing pathway is an important anti-viral defense mechanism in plants. As a counter defense, some members of the viral family Luteoviridae are able to evade host immunity by encoding the P0 RNA silencing suppressor protein. Here we explored the functional diversity of P0 proteins among eight cotton leafroll dwarf virus (CLRDV) isolates, a virus associated with a worldwide cotton disease known as cotton blue disease (CBD).MethodsCLRDV-infected cotton plants of different varieties were collected from five growing fields in Brazil and their P0 sequences compared to three previously obtained isolates. P0’s silencing suppression activities were scored based on transient expression experiments in Nicotiana benthamiana leaves.ResultsHigh sequence diversity was observed among CLRDV P0 proteins, indicating that some isolates found in cotton varieties formerly resistant to CLRDV should be regarded as new genotypes within the species. All tested proteins were able to suppress local and systemic silencing, but with significantly variable degrees. All P0 proteins were able to mediate the decay of ARGONAUTE proteins, a key component of the RNA silencing machinery.ConclusionsThe sequence diversity observed in CLRDV P0s is also reflected in their silencing suppression capabilities. However, the strength of local and systemic silencing suppression was not correlated for some proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0356-7) contains supplementary material, which is available to authorized users.

First genome analysis and molecular characterization of Chickpea chlorotic dwarf virus Egyptian isolate infecting squash.

This study aims to identifying and characterizing some molecular properties of geminiviruses co-infection in squash field crop cultivated in Egypt. Squash crops observed to be heavily infected with several insect vectors, also severe chlorosis and stunting was observed. Electron microscopic analysis has revealed geminate capsid particles which indicate the infection of Geminiviruses, especially SqLCV which represent an economic problem to squash filed crop in Egypt. We have investigated possible mixed infections with different plant viruses associated with chlorotic stunt diseases and or other genus groups of geminiviruses. The main objective of this study is to investigate the recombination events, possible recombinants and variants among these genera in the same family differing in vector transmission. This is the first report of the molecular characterization, phylogenetic analysis and putative recombination events of the full length genome of the Chickpea Chlorotic Dwarf Mastrevirus in Egypt. And the first report of co-infection with another begomovirus infecting squash plants. A full length clone of both viruses were isolated and characterized at the molecular level. The complete nucleotide sequence of DNA-A was determined (2,572bp) and submitted to the genbank under accession no. KF692356. The isolate from Egypt has about 97.8% homology with the Chickpea chlorotic dwarf virus (CpCDV) isolate from Syria DNA-A isolate FR687959, a 83.2% homology with the Sudan isolate AM933134 and a 82.7% homology with Pakistan isolate FR687960. To best of our knowledge this is the first report of complete genome of CpCDV that infect squash plants in Egypt and worldwide.

Genetic diversity and evidence of recombination in the coat protein gene of Onion yellow dwarf virus

The coat protein (CP) coding region of seven Polish Onion yellow dwarf virus (OYDV) isolates was sequenced. It was 771 bp long in all the seven isolates, and encoded a protein of 257 amino acids. Comparative nucleotide sequence analysis revealed diversity of 3 % among the Polish isolates and of 20 % among all garlic isolates worldwide. The comparison of CP sequences of 15 OYDV onion isolates, retrieved from GenBank, revealed an identity of 88–100 and 94–100 % at nt and aa level, respectively. Phylogenetic analysis clustered Polish OYDV isolates in the Subgroup I with isolates from Italy, Argentina and Japan. GARD and SBP algorithms in Datamonkey server and RDP program were employed to detect recombination events in the genome. Recombination analysis provided strong evidence for recombination events in 12 of the sequences analyzed. As regards selection inference, six algorithms (SLAC, FEL, IFEL, REL, MEME and PARRIS) incorporated in HyPhy package were utilized to assess the selection pressure exerted on the expression of OYDV capsid. The ratio of nonsynonymous and synonymous polymorphic sites indicated that purifying selection has acted to eliminate deleterious mutations in coding sites. This is the first report of recombination in OYDV isolates.

Genetic variability among coat protein of Prune dwarf virus variants from different countries and different Prunus species

Prune dwarf virus (PDV) isolates have been investigated for genetic diversity. Full-length nucleotide and amino acid sequences of viral coat protein from 23 isolates collected from different stone fruit trees (sour and sweet cherry trees, wild cherry tree, plum tree, almond tree, peach tree) and different countries (Poland, Italy, Germany, USA, Israel) were analysed and compared to 57 others available in GenBank. Comparison of all sequenced virus isolates revealed diversity of 86–100 % at nucleotide level and 79–100 % at amino acid level. The ratio of non-synonymous to synonymous polymorphic sites indicated that purifying selection dominated in case of PDV. However, six codons showed to be under strong positive selection, including the codon located inside the structure involved in RNA-binding activity.

Sequencing and validation of reference genes to analyze endogenous gene expression and quantify yellow dwarf viruses using RT-qPCR in viruliferous Rhopalosiphum padi.

The bird cherry-oat aphid (Rhopalosiphum padi), an important pest of cereal crops, not only directly sucks sap from plants, but also transmits a number of plant viruses, collectively the yellow dwarf viruses (YDVs). For quantifying changes in gene expression in vector aphids, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a touchstone method, but the selection and validation of housekeeping genes (HKGs) as reference genes to normalize the expression level of endogenous genes of the vector and for exogenous genes of the virus in the aphids is critical to obtaining valid results. Such an assessment has not been done, however, for R. padi and YDVs. Here, we tested three algorithms (GeNorm, NormFinder and BestKeeper) to assess the suitability of candidate reference genes (EF-1α, ACT1, GAPDH, 18S rRNA) in 6 combinations of YDV and vector aphid morph. EF-1α and ACT1 together or in combination with GAPDH or with GAPDH and 18S rRNA could confidently be used to normalize virus titre and expression levels of endogenous genes in winged or wingless R. padi infected with Barley yellow dwarf virus isolates (BYDV)-PAV and BYDV-GAV. The use of only one reference gene, whether the most stably expressed (EF-1α) or the least stably expressed (18S rRNA), was not adequate for obtaining valid relative expression data from the RT-qPCR. Because of discrepancies among values for changes in relative expression obtained using 3 regions of the same gene, different regions of an endogenous aphid gene, including each terminus and the middle, should be analyzed at the same time with RT-qPCR. Our results highlight the necessity of choosing the best reference genes to obtain valid experimental data and provide several HKGs for relative quantification of virus titre in YDV-viruliferous aphids.

A raspberry bushy dwarf virus isolate from Ecuadorean Rubus glaucus contains an additional RNA that is a rearrangement of RNA-2

Sequencing of the complete genome of a raspberry bushy dwarf virus isolate from Rubus glaucus in Ecuador revealed that its RNA-1 and RNA-2 were 5449 and 2231 nucleotides (nt) long, respectively, and phylogenetically closest to isolates from Sweden and Slovenia. In dsRNA analysis of infected plants, an additional band of 3 kbp was observed. Sequencing of this band revealed that it was 3279 nt long. BLAST searches revealed that this band contained a modified version of RNA-2, which consisted of RNA-2 (2231 nt) plus an additional 1048-nt fragment that was concatenated in a reverse-complement fashion to its 5' terminus.

Identifying geographic hot spots of reassortment in a multipartite plant virus.

Reassortment between different species or strains plays a key role in the evolution of multipartite plant viruses and can have important epidemiological implications. Identifying geographic locations where reassortant lineages are most likely to emerge could be a valuable strategy for informing disease management and surveillance efforts. We developed a predictive framework to identify potential geographic hot spots of reassortment based upon spatially explicit analyses of genome constellation diversity. To demonstrate the utility of this approach, we examined spatial variation in the potential for reassortment among Cardamom bushy dwarf virus (CBDV; Nanoviridae, Babuvirus) isolates in Northeast India. Using sequence data corresponding to six discrete genome components for 163 CBDV isolates, a quantitative measure of genome constellation diversity was obtained for locations across the sampling region. Two key areas were identified where viruses with highly distinct genome constellations cocirculate, and these locations were designated as possible geographic hot spots of reassortment, where novel reassortant lineages could emerge. Our study demonstrates that the potential for reassortment can be spatially dependent in multipartite plant viruses and highlights the use of evolutionary analyses to identify locations which could be actively managed to facilitate the prevention of outbreaks involving novel reassortant strains.

Asymmetric patterns of reassortment and concerted evolution in Cardamom bushy dwarf virus

Nanoviruses are single-stranded DNA (ssDNA) plant viruses which have multipartite genomes consisting of discrete, individually encapsidated components. This multipartite strategy may lead to high rates of reassortment, whereby entire genome components are exchanged among different strains. However, few studies have explored the extent to which reassortment shapes the genetic diversity of nanovirus populations. Here we present an extensive analysis of reassortment among 163 Cardamom bushy dwarf virus (CBDV; Nanoviridae family, Babuvirus genus) isolates collected in Northeast India. We also examined evidence of recombination, which is known to play a role in the evolutionary dynamics of nanovirus populations. By sequencing six discrete genome components for each isolate, we demonstrate that over 40% of the isolates display evidence of at least one reassortment event during their evolutionary histories. Nevertheless, a bias in the frequencies at which different genome components reassort was observed, with the DNA-M and DNA-N components being the most predisposed to reassortment. This may reflect variation in the ability of different genome components to function efficiently in a foreign genomic background. Comparisons of the common regions of different genome components revealed signatures of concerted evolution mediated by frequent inter-component homologous recombination. This process, which has previously been reported in nanoviruses and other multipartite ssDNA viruses, may allow proteins which initiate replication to maintain control over distinct genome components. Notably, DNA-N, one of the genome components most prone to reassortment, also exhibited the most frequent inter-component homologous recombination. This supports the idea that inter-component homologous recombination may promote the efficient replication of novel components which are introduced into a genome via reassortment.

Striking differences in the biological and molecular properties of onion and garlic isolates of onion yellow dwarf virus

Complete nucleotide (nt) and deduced amino acid sequences of two onion yellow dwarf virus (OYDV) isolates showing mild and severe symptoms in onion but being unable to infect garlic were determined. The genomes consisted of 10,459 and 10,461 nt (without the 3' poly(A) tail) and were 92.2 % identical. Comparison of their whole genomes, polyproteins and P1, HC-Pro, P3, CI, VPg and NIa-Pro regions with those of garlic isolates previously identified as OYDV gave percentage values below that proposed as the molecular threshold for potyvirus species demarcation. This and the striking differences in host range between onion and garlic isolates suggest that they represent different virus species.