Characterization of a New, Country Bean (Lablab purpureus) Lineage of Bean Common Mosaic Necrosis Virus.

Country bean (Lablab purpureus, family Fabaceae) is grown in subsistence agriculture in Bangladesh as a multipurpose crop for food, animal feed, and green manure. This study was undertaken to investigate the genetic diversity of bean common mosaic necrosis virus (BCMNV, genus Potyvirus, family Potyviridae) in country beans. Leaf samples from country beans showing yellowing, vein banding, and mosaic symptoms were collected during field surveys between 2015 and 2019 cropping seasons from farmers' fields in different geographic regions. These samples were tested by serological and molecular diagnostic assays for the presence of BCMNV. Virus-positive samples were subjected to high-throughput Illumina sequencing to generate near-complete genomes of BCMNV isolates. In pairwise comparisons, the polyprotein sequences of BCMNV isolates from Bangladesh showed greater than 98% identities among themselves and shared less than 84% sequence identity at the nucleotide level with virus isolates reported from other countries. In the phylogenetic analysis, BCMNV isolates from Bangladeshi country beans formed a separate clade from virus isolates reported from common beans in other countries in the Americas, Africa, Europe, and from East Timor. Grow-out studies showed seed-to-seedling transmission of BCMNV, implying a possible seedborne nature of the virus in country beans.

Molecular characterization of a novel gammacarmovirus infecting cucurbits in India.

In this study, we describe the identification of a new gammacarmovirus infecting Cucurbita pepo plants showing a range of mosaic, stunting, yellowing, and wilting symptoms. The virus had a narrow host range and mostly produced chlorotic and necrotic local lesions in the majority of the tested plants. However, Nicotiana benthamiana showed systemic symptoms under laboratory conditions. Using a combination of Sanger sequencing and rapid amplification of cDNA ends (RACE), the complete genome sequence of the virus was determined to be 4274 nucleotides (nt) in length. Its genome organization is similar to that of members of the genus Gammacarmovirus in the family Tombusviridae, consisting of five overlapping open reading frames (ORFs) encoding p28, replicase, p7A, p7B, and coat protein (CP), respectively. The genome is flanked by short 5' and 3' non-coding regions (NCR) at either end. In pairwise comparisons of replicase and CP sequences, the virus showed the highest amino acid sequence identity of 71.55% and 54.86%, respectively, to melon necrotic spot virus (MNSV), the type member of the genus Gammacarmovirus. Since the sequence identity values are below the species demarcation threshold suggested by the International Committee on Taxonomy of Viruses (ICTV), the virus from Cucurbita pepo plants, for which the name "cucurbit carmovirus" (CuCV) is proposed, represents a new species. In phylogenetic analysis based on the replicase and CP amino acid sequences, CuCV clustered with MNSV but formed a distinct branch, further confirming that the virus is a distinct member of the genus Gammacarmovirus.

Spatio-Temporal Spread of Grapevine Leafroll Disease in Washington State Vineyards.

The spread of grapevine leafroll disease (GLD) to vineyards planted with certified planting stock is of significant concern to grape growers. In this study, the spatial and temporal spread of GLD was examined in three vineyard blocks planted with virus-tested wine grape (Vitis vinifera) cultivars adjacent to vineyard blocks heavily infected with GLD in two geographic locations in eastern Washington State. During each season, the position of vines showing GLD symptoms was recorded in a matrix representing the planting lattice. Symptomatic vines were positive only for Grapevine leafroll-associated virus 3 (GLRaV-3), the most common virus species consistently associated with GLD in Washington vineyards. The results from multiple seasons showed a gradual increase in disease incidence over time in all three blocks. Spatial and temporal mapping of GLD indicated a disease gradient in which the highest percentage of symptomatic vines was in rows proximal to infected old blocks. Spatial autocorrelation analysis using Moran's I values suggested random patterns of symptomatic vines in the three blocks during initial years, indicating primary spread of the virus not related to infected vines within the block. Clustering at the scale of neighboring vines during subsequent years suggested secondary spread within the block. Results of quadrat-based spatial analyses of GLD incidence were compared with previously reported data obtained from California and elsewhere for an improved understanding of the dynamics of GLD spread to facilitate area-wide disease management strategies.

Intra-species recombination among strains of the ampelovirus Grapevine leafroll-associated virus 4.

BACKGROUND: Grapevine leafroll disease is one of the most economically important viral diseases affecting grape production worldwide. Grapevine leafroll-associated virus 4 (GLRaV-4, genus Ampelovirus, family Closteroviridae) is one of the six GLRaV species documented in grapevines (Vitis spp.). GLRaV-4 is made up of several distinct strains that were previously considered as putative species. Currently known strains of GLRaV-4 stand apart from other GLRaV species in lacking the minor coat protein. METHODS: In this study, the complete genome sequence of three strains of GLRaV-4 from Washington State vineyards was determined using a combination of high-throughput sequencing, Sanger sequencing and RACE. The genome sequence of these three strains was compared with corresponding sequences of GLRaV-4 strains reported from other grapevine-growing regions. Phylogenetic analysis and SimPlot and Recombination Detection Program (RDP) were used to identify putative recombination events among GLRaV-4 strains. RESULTS: The genome size of GLRaV-4 strain 4 (isolate WAMR-4), strain 5 (isolate WASB-5) and strain 9 (isolate WALA-9) from Washington State vineyards was determined to be 13,824 nucleotides (nt), 13,820 nt, and 13,850 nt, respectively. Multiple sequence alignments showed that a 11-nt sequence (5'-GTAATCTTTTG-3') towards 5' terminus of the 5' non-translated region (NTR) and a 10-nt sequence (5'-ATCCAGGACC-3') towards 3' end of the 3' NTR are conserved among the currently known GLRaV-4 strains. LR-106 isolate of strain 4 and Estellat isolate of strain 6 were identified as recombinants due to putative recombination events involving divergent sequences in the ORF1a from strain 5 and strain Pr. CONCLUSION: Genome-wide analyses showed for the first time that recombinantion can occur between distinct strains of GLRaV-4 resulting in the emergence of genetically stable and biologically successful chimeric viruses. Although the origin of recombinant strains of GLRaV-4 remains elusive, intra-species recombination could be playing an important role in shaping genetic diversity and evolution of the virus and modulating the biology and epidemiology of GLRaV-4 strains.

Sequence analysis of the medium and small RNAs of impatiens necrotic spot virus reveals segment reassortment but not recombination.

The complete sequence of the medium (M) and small (S) RNA genome segments were determined for twelve isolates of impatiens necrotic spot virus from eight plant species. The M- and S-RNAs of these isolates shared 97-99% and 93-98% nucleotide sequence identity, respectively, with the corresponding full-length sequences available in public databases. Phylogenetic analysis based on the M- or S-RNA sequences showed incongruence in the phylogenetic position of some isolates, suggesting intraspecies segment reassortment. The lack of phylogenetic discordance in individual and concatenated sequences of individual genes encoded by M- or S-RNAs suggests that segment reassortment rather than recombination is driving evolution of these INSV isolates.

Integrating Local Lesion Assays with Conventional RT-PCR for Detection of Interspecies Tospovirus Reassortants and Mixed Tospovirus Infections.

Tomato spotted wilt virus (TSWV) has historically been the major tospovirus present in North America. Recent emergence of Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV) in Florida and the Caribbean has complicated reliable identification of tospoviruses in this region. Field symptoms of these three tospoviruses are indistinguishable in most host plants, and commercially available TSWV lateral-flow immunoassay reagents cross react with GRSV and TCSV, leading to incorrect diagnoses of GRSV or TCSV as TSWV. Reliable diagnosis of TSWV, GRSV, and TCSV is further confounded by the fact that all currently known isolates of GRSV in the United States are reassortants containing one genomic RNA segment derived from TCSV. To address these practical challenges, we developed and validated genome segment-specific primers for conventional reverse-transcription polymerase chain reaction (RT-PCR) detection of the large, medium, and small RNA segments of TSWV, GRSV, and TCSV. When used in conjunction with local lesion-passaged virus isolates, the genome segment-specific RT-PCR assays developed in this study will facilitate high-throughput screening of plant or thrips samples for interspecies reassortants in epidemiological studies and reliable identification of these three tospoviruses in mixed infections commonly observed in the field.

The Relative Occurrence of Grapevine leafroll-associated virus 3 and Grapevine red blotch virus in Washington State Vineyards.

Vineyard surveys were conducted for three consecutive seasons in eastern Washington State, the major grapevine-growing region in the state, to document the occurrence of Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine red blotch virus (GRBV). The majority of samples were collected from red-berried wine grape (Vitis vinifera) cultivars exhibiting symptoms of or suspected for grapevine leafroll (GLD) and red blotch (GRBD) diseases. A limited number of samples from white-berried cultivars were collected randomly due to the lack of visual symptoms. Samples were collected from a total of 2,063 grapevines from 18 red-berried cultivars and seven white-berried cultivars planted in eight American Viticultural Areas and tested for GLRaV-3 and GRBV using RT-PCR and PCR, respectively. The results showed 67.77% and 6.01% of total samples positive for GLRaV-3 and GRBV, respectively, and 9.06% of samples positive for both viruses. About 17% of samples tested negative for the two viruses, but some of these samples were positive for GLRaV-2 and GLRaV-4. Overall results indicated that GLRaV-3 was more common than GRBV, independent of cultivars and the geographic origin of samples. Due to variability in symptoms in red-berried cultivars, virus-specific diagnostic assays were deemed necessary for reliable identification of GLRaV-3 and GRBV and to differentiate GLD and GRBD symptoms from those induced by biotic and abiotic stresses in vineyards. A multiplex PCR protocol was developed for simultaneous detection of GLRaV-3 and GRBV in grapevine samples. A global phylogenetic analysis of GRBV genome sequences revealed segregation of virus isolates from Washington State vineyards into two distinct clades, with the majority of isolates belonging to clade II.

An Analysis of the Complete Genome Sequence and Subgenomic RNAs Reveals Unique Features of the Ampelovirus, Grapevine leafroll-associated virus 1.

Despite being the first closterovirus documented in grapevines (Vitis sp.), the molecular biology of Grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus, family Closteroviridae) is still in its infancy. In this study, the complete genome sequence of two GLRaV-1 isolates was determined to be 18,731 (isolate WA-CH) and 18,946 (isolate WA-PN) nucleotides (nt). The genome of WA-CH and WA-PN isolates encodes nine putative open reading frames (ORFs) and the arrangement of these ORFs in both isolates was similar to that of Australian and Canadian isolates. In addition to two divergent copies of the coat protein (CP), the genome of GLRaV-1 isolates contain CP-homologous domain in four genes, making the virus unique among Closteroviridae members. The 5' and 3' nontranslated regions (NTRs) of WA-CH and WA-PN isolates showed differences in size and sequence composition, with 5' NTR having variable number of ∼65-nt-long repeats. Using the 5' NTR sequences, a reverse transcription-polymerase chain reaction and restriction fragment length polymorphism method was developed to distinguish GLRaV-1 variants in vineyards. Northern analysis of total RNA from GLRaV-1-infected grapevine samples revealed three subgenomic RNAs (sgRNAs), corresponding tentatively to CP, p21, and p24 ORFs, present at higher levels, with p24 sgRNA observed at relatively higher abundance than the other two sgRNAs. The 5' terminus of sgRNAs corresponding to CP, CPd1, CPd2, p21, and p24 were mapped to the virus genome and the leader sequence for these five sgRNAs determined to be 68, 27, 15, 49, and 18 nt, respectively. Taken together, this study provided a foundation for further elucidation of the comparative molecular biology of closteroviruses infecting grapevines.

Nicotiana benthamiana: Its History and Future as a Model for Plant-Pathogen Interactions.

Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Addi- tionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host-pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virus- induced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, inter- action, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions cur- rently used by the research community. In addition to ad- dressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.

First Report of Cucumber mosaic virus from Eggplant (Solanum melongena) in Bangladesh.

In Bangladesh, eggplant (Solanum melongena L.) is largely cultivated by subsistence farmers for domestic consumption and generating family income. During a survey of family-owned farms in April of 2014 in Barisal region of Bangladesh, we observed a farmer's field (0.25 acres) of 3-month-old eggplants with nearly 90% of plants showing mild mosaic and mottling of leaves. Symptomatic plants showed reduced growth, with nearly 50% fewer fruits than from healthy plants. Symptomatic leaves tested positive for Cucumber mosaic virus (CMV; genus Cucumovirus, family: Bromoviridae) by immunostrip diagnostic kit (Agdia, Elkhart, IN). For confirmation of the virus identity, leaf samples were pressed on FTA Plant Cards (Whatman International, Maidstone, UK) and air-dried at room temperature. For eluting total nucleic acids, four to eight disks were punched from the spotted circles of each FTA card using a Harris micropunch (2-mm diameter, Sigma-Aldrich, USA) and soaked for 1 h in 300 µl of extraction buffer (15 mM Na2CO3, 35 mM NaHCO3, 2% [w/v] PVP40, 0.2% [w/v] BSA, 0.05% [v/v] Tween 20, pH 9.6). After vortexing followed by a brief centrifugation, 10 µl of the supernatant was mixed with denaturing buffer (0.1M glycine-NaOH, pH 9.0, 50 mM NaCl, 1 mM EDTA, pH 8.0, 0.5% [v/v] Triton X-100) containing 1% ß-mercaptoethanol, incubated at 95°C for 10 min, and kept in ice until use. Denatured sample (2 µl) was subsequently used in reverse-transcription (RT)-PCR using primers CMV-RNA3F (5'-GTAGACATCTGTGACGCGA-3') and CMV-RNA3R (5'-GCGCGAAACAAGCTTCTTATC-3') previously reported (2) to amplify a 529-nucleotide (nt) fragment representing the 210-nt intergenic region and the 319-nt partial coat protein (CP) gene of the RNA 3 segment. The amplicons were cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA), and DNA isolated from four independent clones per amplicon was sequenced in both orientations. The derived sequences (GenBank Accession Nos. KM516898 to KM516901) showed close to 100% identity among themselves and 97% identity with the corresponding sequence of CMV isolate BK16 from cucumber in Thailand (FN552546). These results supported immunostrip diagnostic assays in confirming the presence of CMV in symptomatic samples of eggplants from Barisal. For additional confirmation, a second primer pair (CMV-CP-F: 5'-ATGGACAAATCTGAATCAACCAG-3' and CMV-CP-R: 5'-TCAAACTGGGAGCACCCCAGAC-3') was designed using CMV sequences from JN054635 and GU906293 to amplify the full-length CP gene from the same nucleic acid preparations used above. The approximately 657-nt amplicons, representing the full-length CP gene, were cloned, and plasmid DNA from four independent colonies per amplicon wa s sequenced as described above. The derived CP sequences (KM516902 to KM516905) shared 96 and 95% nucleotide and 98.6 and 99.5% amino acid sequence identities with corresponding sequences of CMV isolates from banana (EF178298) and eggplant (GU906293), respectively, from India. Phylogenetic analysis of CP sequences derived from this study with corresponding sequences available in GenBank indicated that CMV from eggplant in Bangladesh aligned closely with CMV subgroup 1B. CMV was previously reported in chili pepper, and tomato from Bangladesh (1) and in eggplant from Israel (4) and India (3). To our knowledge this is the first confirmed report of the occurrence of CMV subgroup 1B in eggplant in Bangladesh. Since no aphids were observed on eggplants, it is likely that CMV was introduced into the farmer's field through seedlings raised from seed carrying the virus. References: (1) A. M. Akanda et al. J. Fac. Agric., Kyushu Univ. 35:151, 1991. (2) C. De Blas et al. J. Phytopathol. 141:323, 1994. (3) S. Kumar et al. Virus Dis. 25:129, 2014. (4) E. Tanne and S. Zimmerman-Gries. Plant Dis. 64:371, 1980.

Genetic diversity of Grapevine virus A in Washington and California vineyards.

Grapevine virus A (GVA; genus Vitivirus, family Betaflexiviridae) has been implicated with the Kober stem grooving disorder of the rugose wood disease complex. In this study, 26 isolates of GVA recovered from wine grape (Vitis vinifera) cultivars from California and Washington were analyzed for their genetic diversity. An analysis of a portion of the RNA-dependent RNA polymerase (RdRp) and complete coat protein (CP) sequences revealed intra- and inter-isolate sequence diversity. Our results indicated that both RdRp and CP are under strong negative selection based on the normalized values for the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site. A global phylogenetic analysis of CP sequences revealed segregation of virus isolates into four major clades with no geographic clustering. In contrast, the RdRp-based phylogenetic tree indicated segregation of GVA isolates from California and Washington into six clades, independent of geographic origin or cultivar. Phylogenetic network coupled with recombination analyses showed putative recombination events in both RdRp and CP sequence data sets, with more of these events located in the CP sequence. The preponderance of divergent variants of GVA co-replicating within individual grapevines could increase viral genotypic complexity with implications for phylogenetic analysis and evolutionary history of the virus. The knowledge of genetic diversity of GVA generated in this study will provide a foundation for elucidating the epidemiological characteristics of virus populations at different scales and implementing appropriate management strategies for minimizing the spread of genetic variants of the virus by vectors and via planting materials supplied to nurseries and grape growers.

Molecular characterization and impacts of a strain of Grapevine leafroll-associated virus 2 causing asymptomatic infection in a wine grape cultivar.

BACKGROUND: Grapevine leafroll (GLD) is considered as the most economically important virus disease affecting wine grapes (Vitis vinifera L.) in many grapevine-growing regions. GLD produces distinct symptoms in red- and white-berried cultivars. In this study, we determined the complete genome sequence of an asymptomatic strain of Grapevine leafroll-associated virus 2 (GLRaV-2) and studied its impacts on fruit yield and berry quality attributes in an own-rooted, red-berried wine grape cultivar. FINDINGS: The complete genome of GLRaV-2 obtained from a red-berried wine grape cultivar Sangiovese, designated as GLRaV-2-SG, was determined to be 16,474 nucleotides in length. In pairwise comparisons, using complete genome sequences of GLRaV-2 strains available in GenBank, GLRaV-2-SG was more closely related to GLRaV-2-OR1 from Oregon, USA, and GLRaV-2-93/955 from South Africa, and distantly related to GLRaV-2-BD from Italy and GLRaV-2-RG from USA. Fruit yield estimates and berry quality analysis at the time of commercial harvest indicated that GLRaV-2-SG had little impact on fruit yield and total soluble solids, juice pH and total anthocyanins of berry skin. CONCLUSIONS: Because so little is known about the effects of asymptomatic virus infections in wine grapes, this study expanded our knowledge of the occurrence and impacts of GLRaV-2 causing asymptomatic infections. Our results indicated that an asymptomatic strain of GLRaV-2 may not cause significant effects to overall fruit yield and berry quality in own-rooted vines, but can affect its host in more subtle ways. Since disease symptoms are not apparent, relying on visual symptoms during disease surveys may result in the escape of asymptomatic strains of GLRaV-2. Thus, it is necessary to use appropriate diagnostic assays for reliable detection of viruses causing asymptomatic infections.

Complete genome sequence analysis of an American isolate of Grapevine virus E.

The complete genome sequence of Grapevine virus E (GVE) collected from a red-berried wine grape cultivar (Cabernet Sauvignon) in Washington State was determined. The 7,568 nucleotide long genome of GVE is more similar in sequence identity with a GVE isolate from a wine grape cv. Shiraz from South Africa when compared with an isolate from "Aki Queen" grape from Japan. Like GVE isolates from South Africa and Japan, the Washington isolate encodes five open reading frames (ORFs) and the overall genome organization is identical among these isolates. In addition to AlkB domain, a DExD domain, belonging to the DEAD-like helicases superfamily, was present upstream of the helicase domain in the replicase ORF of the virus.

Grapevine leafroll-associated virus 1 occurs as genetically diverse populations.

The genetic diversity of 34 isolates of Grapevine leafroll-associated virus 1 (GLRaV-1) from different wine, table, and ornamental grape cultivars in California, New York, and Washington States in the United States was investigated. Segments of the heat-shock protein 70 homolog (HSP70h) gene, coat protein (CP) gene, coat protein duplicate 2 (CPd2) gene, and open reading frame 9 (p24) were amplified by reverse-transcription polymerase chain reaction, cloned, and sequenced. A pairwise comparison of nucleotide sequences revealed intra- and interisolate sequence diversity, with CPd2 and HSP70h being the most and the least divergent, respectively, among the four genomic regions studied. The normalized values for the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site indicated different purifying selection pressures acting on each of the four genomic regions, with the CP and CPd2 being subjected to the strongest and weakest functional constraints, respectively. A global phylogenetic analysis of sequences from the four genomic regions revealed segregation of GLRaV-1 isolates into three major clades and a lack of clearly defined clustering by geographical origin. In contrast, only two lineages were apparent when the CP and CPd2 gene sequences were used in phylogenetic analyses. Putative recombination events were revealed among the HSP70h, CP, and p24 sequences. The genetic landscape of GLRaV-1 populations presented in this study provides a foundation for better understanding of the epidemiology of grapevine leafroll disease across grape-growing regions in the United States. In addition, this study will benefit grape clean plant programs across the country in improving the sanitary status of planting materials provided to nurseries and grape growers.

Importance and genetic diversity of vegetable-infecting tospoviruses in India.

A survey for Peanut bud necrosis virus (PBNV), Watermelon bud necrosis virus (WBNV), Capsicum chlorosis virus (CaCV), and Iris yellow spot virus (IYSV) was conducted between 2002 and 2009 in the major vegetable-growing areas in India. PBNV was documented widely in tomato and chili peppers in 14 states representing southern, north-western, north-eastern, and central regions and WBNV was predominantly detected in watermelons and cucurbits in all except north-eastern regions. In addition, the expanded host range of PBNV to watermelons and other cucurbits and WBNV to tomato and chili peppers was observed leading to natural mixed infection of the two viruses. IYSV was found in onion in southern, central, and north-eastern regions and CaCV in tomato and chili peppers in northern and southern regions, respectively. Phylogenetic analysis of the nucleocapsid gene revealed segregation of field isolates of PBNV and WBNV into two distinct subclades, whereas isolates of CaCV and IYSV each clustered into a single clade. A proposal for establishing WBNV as a distinct tospovirus species is made based on the molecular characterization of small- (S) and medium- (M) RNA segments.

Sequence diversity, population genetics and potential recombination events in grapevine rupestris stem pitting-associated virus in Pacific North-West vineyards.

Grapevine rupestris stem pitting-associated virus (GRSPaV; genus Foveavirus, family Flexiviridae) is present in many grape-growing regions of the world. A total of 84 full-length coat protein (CP) sequences and 57 sequences representing the helicase-encoding region (HR) of the RNA-dependent RNA polymerase were obtained from wine grape cultivars grown in the Pacific North-West (PNW) of the United States and their molecular diversity was compared with corresponding sequences previously reported from other grape-growing regions. In pairwise comparisons, the CP sequences from PNW isolates showed identities between 80 and 100% at the nucleotide level and the HR sequences showed identities between 79 and 100%. A global phylogenetic analysis of the CP and HR sequences revealed segregation of GRSPaV isolates into four major lineages with isolates from PNW distributed in all four lineages, indicating a lack of clustering by geographical origin. Scion cultivars grafted onto rootstock were found to contain mixtures of more genetic variants belonging to different lineages than own-rooted cultivars. Assessment of population genetic parameters found that the CP was more variable than the HR region. The discordant gene phylogenies obtained for some CP and HR sequences and the identification of potential recombination events involving parents from different lineages provided strong evolutionary evidence for genetic diversity among GRSPaV isolates. These results underscore the highly variable nature of the virus with implications for grapevine health status and distribution of virus-tested planting materials. This study also contributes to an increased understanding of molecular population genetics of viruses infecting deciduous woody perennials.

Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves.

BACKGROUND: Symptoms of grapevine leafroll disease (GLRD) in red-fruited wine grape (Vitis vinifera L.) cultivars consist of green veins and red and reddish-purple discoloration of inter-veinal areas of leaves. The reddish-purple color of symptomatic leaves may be due to the accumulation of anthocyanins and could reflect an up-regulation of genes involved in their biosynthesis. RESULTS: We examined six putative constitutively expressed genes, Ubiquitin, Actin, GAPDH, EF1-a, SAND and NAD5, for their potential as references for normalization of gene expression in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Using the geNorm program, a combination of two genes (Actin and NAD5) was identified as the stable set of reference genes for normalization of gene expression data obtained from grapevine leaves. By using gene-specific RT-qPCR in combination with a reliable normalization factor, we compared relative expression of the flavonoid biosynthetic pathway genes between leaves infected with Grapevine leafroll-associated virus 3 (GLRaV-3) and exhibiting GLRD symptoms and virus-free green leaves obtained from a red-fruited wine grape cultivar (cv. Merlot). The expression levels of these different genes ranged from two- to fifty-fold increase in virus-infected leaves. Among them, CHS3, F3'5'H, F3H1, LDOX, LAR1 and MybA1 showed greater than 10-fold increase suggesting that they were expressed at significantly higher levels in virus-infected symptomatic leaves. HPLC profiling of anthocyanins extracted from leaves indicated the presence of cyanidin-3-glucoside and malvidin-3-glucoside only in virus-infected symptomatic leaves. The results also showed 24% higher levels of flavonols in virus-infected symptomatic leaves than in virus-free green leaves, with quercetin followed by myricetin being the predominant compounds. Proanthocyanidins, estimated as total tannins by protein precipitation method, were 36% higher in virus-infected symptomatic leaves when compared to virus-free green leaves. CONCLUSIONS: The results, the first example to our knowledge, showed that modulation of the flavonoid biosynthetic pathway occurred in GLRaV-3-infected leaves of a red-fruited wine grape cultivar (cv. Merlot) leading to de novo synthesis of two classes of anthocyanins. These anthocyanins have contributed to the expression of reddish-purple color of virus-infected grapevine leaves exhibiting GLRD symptoms.

3'-coterminal subgenomic RNAs and putative cis-acting elements of Grapevine leafroll-associated virus 3 reveals 'unique' features of gene expression strategy in the genus Ampelovirus.

BACKGROUND: The family Closteroviridae comprises genera with monopartite genomes, Closterovirus and Ampelovirus, and with bipartite and tripartite genomes, Crinivirus. By contrast to closteroviruses in the genera Closterovirus and Crinivirus, much less is known about the molecular biology of viruses in the genus Ampelovirus, although they cause serious diseases in agriculturally important perennial crops like grapevines, pineapple, cherries and plums. RESULTS: The gene expression and cis-acting elements of Grapevine leafroll-associated virus 3 (GLRaV-3; genus Ampelovirus) was examined and compared to that of other members of the family Closteroviridae. Six putative 3'-coterminal subgenomic (sg) RNAs were abundantly present in grapevine (Vitis vinifera) infected with GLRaV-3. The sgRNAs for coat protein (CP), p21, p20A and p20B were confirmed using gene-specific riboprobes in Northern blot analysis. The 5'-termini of sgRNAs specific to CP, p21, p20A and p20B were mapped in the 18,498 nucleotide (nt) virus genome and their leader sequences determined to be 48, 23, 95 and 125 nt, respectively. No conserved motifs were found around the transcription start site or in the leader sequence of these sgRNAs. The predicted secondary structure analysis of sequences around the start site failed to reveal any conserved motifs among the four sgRNAs. The GLRaV-3 isolate from Washington had a 737 nt long 5' nontranslated region (NTR) with a tandem repeat of 65 nt sequence and differed in sequence and predicted secondary structure with a South Africa isolate. Comparison of the dissimilar sequences of the 5'NTRs did not reveal any common predicted structures. The 3'NTR was shorter and more conserved. The lack of similarity among the cis-acting elements of the diverse viruses in the family Closteroviridae is another measure of the complexity of their evolution. CONCLUSIONS: The results indicate that transcription regulation of GLRaV-3 sgRNAs appears to be different from members of the genus Closterovirus. An analysis of the genome sequence confirmed that GLRaV-3 has an unusually long 5'NTR of 737 nt compared to other monopartite members of the family Closteroviridae, with distinct differences in the sequence and predicted secondary structure when compared to the corresponding region of the GLRaV-3 isolate from South Africa.

Two new 'legumoviruses' (genus Begomovirus) naturally infecting soybean in Nigeria.

Two new 'legumoviruses' (genus Begomovirus; family Geminiviridae) naturally infecting soybean (Glycine max L. Merr.) in Nigeria were molecularly characterized. Based on characteristic symptoms in soybean, the two viruses are provisionally designated as Soybean mild mottle virus (SbMMV) and Soybean chlorotic blotch virus (SbCBV). SbCBV has a bipartite genome, whereas SbMMV has only a DNA A component. The DNA A component of SbMMV is 2,768 nucleotides (nt) long and the DNA A and DNA B components of SbCBV are 2,708 and 2,647 nt long, respectively. In pairwise comparisons, the DNA A component of SbMMV and SbCBV showed 62% nt sequence identity, indicating that these two viruses are distinct. Whereas the DNA A of SbMMV contains two virion- and four complementary-sense open reading frames, that of SbCBV lacks the virus-sense AV2, a signature gene present in 'Old World' begomoviruses. A pairwise comparison with the corresponding nucleotide sequence of other begomoviruses in the databases indicated that SbCBV had a maximum of 74% identity with cowpea golden mosaic virus and SbMMV had a maximum of 65% identity with mungbean yellow mosaic India virus and kudzu mosaic virus. Phylogenetic analysis of the DNA A component of SbCBV and SbMMV together with those of other begomoviruses available in the databases showed clustering of the two viruses within the 'legumovirus' clade of the begomovirus phylogenetic tree. In addition, the DNA A and B components of SbCBV from Centrosema pubescens Benth were found to be identical to those from soybean, indicating that leguminous wild species are a potential alternative host for the virus. Since soybean is an introduced crop, the identification of two distinct begomoviruses naturally infecting soybean in Nigeria suggests the occurrence of 'legumoviruses' in plant species indigenous to Africa and underscores their potential threat to sustainable cultivation of soybean on the African continent.

Genetic variability of natural populations of Grapevine leafroll-associated virus 2 in Pacific Northwest vineyards.

Genetic variability of field populations of Grapevine leafroll-associated virus 2 (GLRaV-2) in Pacific Northwest (PNW) vineyards was characterized by sequencing the entire coat protein (CP) and a portion of the heat-shock protein-70 homolog (HSP70h) genes. Phylogenetic analysis of CP and HSP70h nucleotide sequences obtained in this study and corresponding sequences from GenBank revealed segregation of GLRaV-2 isolates into six lineages with virus isolates from PNW distributed in 'PN', 'H4', and 'RG' lineages. An estimation of the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site indicated that different selection pressures may be acting on the two genomic regions encoding proteins with distinct functions. Multiple alignments of CP amino acid sequences showed lineage-specific differences. Enzyme-linked immunosorbent assay results indicated that GLRaV-2-specific antibodies from a commercial source are unable to reliably detect GLRaV-2 isolates in the RG lineage, thereby limiting antibody-based diagnosis of all GLRaV-2 isolates currently found in PNW vineyards. A protocol based on reverse-transcription polymerase chain reaction and restriction fragment length polymorphism analysis was developed for differentiating GLRaV-2 isolates belonging to the three lineages present in the region. The taxonomic status of GLRaV-2 is discussed in light of the current knowledge of global genetic diversity of the virus.