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The cultivation of pineapple (Ananas comosus) is threatened worldwide by mealybug wilt disease of pineapple (MWP), whose etiology is not yet fully elucidated. In this study, we characterized pineapple mealybug wilt-associated ampeloviruses (PMWaVs, family Closteroviridae) from a diseased pineapple plant collected from Reunion Island, using a high-throughput sequencing approach combining Illumina short reads and Nanopore long reads. Reads co-assembly resulted in complete or near-complete genomes for six distinct ampeloviruses, including the first complete genome of pineapple mealybug wilt-associated virus 5 (PMWaV5) and that of a new species tentatively named pineapple mealybug wilt-associated virus 7 (PMWaV7). Short reads data provided high genome coverage and sequencing depths for all six viral genomes, contrary to long reads data. The 5′ and 3′ ends of the genome for most of the six ampeloviruses could be recovered from long reads, providing an alternative to RACE-PCRs. Phylogenetic analyses did not unveil any geographic structuring of the diversity of PMWaV1, PMWaV2 and PMWaV3 isolates, supporting the current hypothesis that PMWaVs were mainly spread by human activity and vegetative propagation. [ABSTRACT FROM AUTHOR]

The cultivation of pineapple (Ananas comosus) is threatened worldwide by mealybug wilt disease of pineapple (MWP), whose etiology is not yet fully elucidated. In this study, we characterized pineapple mealybug wilt-associated ampeloviruses (PMWaVs, family Closteroviridae) from a diseased pineapple plant collected from Reunion Island, using a high-throughput sequencing approach combining Illumina short reads and Nanopore long reads. Reads co-assembly resulted in complete or near-complete genomes for six distinct ampeloviruses, including the first complete genome of pineapple mealybug wilt-associated virus 5 (PMWaV5) and that of a new species tentatively named pineapple mealybug wilt-associated virus 7 (PMWaV7). Short reads data provided high genome coverage and sequencing depths for all six viral genomes, contrary to long reads data. The 5′ and 3′ ends of the genome for most of the six ampeloviruses could be recovered from long reads, providing an alternative to RACE-PCRs. Phylogenetic analyses did not unveil any geographic structuring of the diversity of PMWaV1, PMWaV2 and PMWaV3 isolates, supporting the current hypothesis that PMWaVs were mainly spread by human activity and vegetative propagation.

This article reports on the discovery of three pineapple mealybug wilt-associated viruses (PMWaVs) causing pineapple mealybug wilt disease in East Malaysia, specifically in Sarawak, Borneo. Pineapple farmers in Sarawak have been observing widespread symptoms of mealybug wilt since 2021, including wilting, leaf curl, and discoloration. The researchers collected samples from different pineapple plantations in Sarawak and confirmed the presence of PMWaV-1, -2, and -3 through molecular analysis. This study provides important insights into the epidemiology of PMWaV in Sarawak and can inform disease management strategies to protect pineapple production in East Malaysia. [Extracted from the article]

Cassava Brown Streak Disease (CBSD), which is caused by cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), represents one of the most devastating threats to cassava production in Africa, including in Rwanda where a dramatic epidemic in 2014 dropped cassava yield from 3.3 million to 900,000 tonnes (1). Studying viral genetic diversity at the genome level is essential in disease management, as it can provide valuable information on the origin and dynamics of epidemic events. To fill the current lack of genome-based diversity studies of UCBSV, we performed a nationwide survey of cassava ipomovirus genomic sequences in Rwanda by high-throughput sequencing (HTS) of pools of plants sampled from 130 cassava fields in thirteen cassava-producing districts, spanning seven agro-ecological zones with contrasting climatic conditions and different cassava cultivars. HTS allowed the assembly of a nearly complete consensus genome of UCBSV in twelve districts. The phylogenetic analysis revealed high homology between UCBSV genome sequences, with a maximum of 0.8 per cent divergence between genomes at the nucleotide level. An in-depth investigation based on Single Nucleotide Polymorphisms (SNPs) was conducted to explore the genome diversity beyond the consensus sequences. First, to ensure the validity of the result, a panel of SNPs was confirmed by independent reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Furthermore, the combination of fixation index (F ST) calculation and Principal Component Analysis (PCA) based on SNP patterns identified three different UCBSV haplotypes geographically clustered. The haplotype 2 (H2) was restricted to the central regions, where the NAROCAS 1 cultivar is predominantly farmed. RT-PCR and Sanger sequencing of individual NAROCAS1 plants confirmed their association with H2. Haplotype 1 was widely spread, with a 100 per cent occurrence in the Eastern region, while Haplotype 3 was only found in the Western region. These haplotypes' associations with specific cultivars or regions would need further confirmation. Our results prove that a much more complex picture of genetic diversity can be deciphered beyond the consensus sequences, with practical implications on virus epidemiology, evolution, and disease management. Our methodology proposes a high-resolution analysis of genome diversity beyond the consensus between and within samples. It can be used at various scales, from individual plants to pooled samples of virus-infected plants. Our findings also showed how subtle genetic differences could be informative on the potential impact of agricultural practices, as the presence and frequency of a virus haplotype could be correlated with the dissemination and adoption of improved cultivars. [ABSTRACT FROM AUTHOR]

This research studied serological relationships and genetic diversity of Argentinean isolates of grapevine leafroll-associated virus-4 (GLRaV-4). Phylogenetic analysis of coat protein (CP) sequences from 19 local isolates revealed clustering with the previously described GLRaV-4 strain 5, strain 6, and strain 9 groups. Evolutionary sequence analysis of the obtained and database-available sequences showed evidence of recombination events. Additionally, both CP N- and C-terminal regions appeared to be under purifying selection, but the N-terminal region presented seven sites under positive selection, with a dN/dS ratio 5-fold greater than that of the C-terminal region. Serological reactivity against monoclonal antibodies supports a higher occurrence probability for linear epitopes in the N-terminal region, as inferred by the sequence analysis. The obtained results reflect an unusual evolutionary behavior of the CP that, together with protein serological reactivity, suggests biological significance of the observed variability. Highlights: • The occurrence of GLRaV-4 strains -5, -6 and -9 was recorded in Argentina. • The coat protein sequence showed a highly variable N-terminal region among the strains. • An evolutionary analysis inferred the occurrence of sites under positive diversifying selection pressure. • The most variable region of the coat protein presented the highest probability of occurrence of linear epitopes.

Biotic and abiotic stress seriously affects the development of plants, leading to the death of a significant number of plants in natural landscapes. Over the last twelve years, the central zone of Chile has been under an intensive drought, affecting the species inhabiting the Chilean Mediterranean forest, which is classified as a biological hotspot. In this context, our group started a small survey to monitor the presence of intracellular pathogens that may be infecting the plants, increasing the damage caused by the water deprivation. Several plants of one species, Persea lingue, showed symptoms of interveinal yellowing and leaf curling. RNA-seq analyses of one of these samples showed the presence of a long contig with high coverage only in symptomatic plants. Phylogenetic analysis and the molecular features indicated that it was a new species of the Ampelovirus genus. RT-PCR analyses determined the presence of the virus only in symptomatic plants of the same natural preserve. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease is one of the most important virus diseases of grapevines and occurs in every major grape-growing region of the world. The vector-transmission mechanisms of the causative agent, Grapevine leafroll-associated virus 3 (GLRaV-3), remain poorly understood. We show that the vine mealybug, Planococcus ficus, feeds through a membrane feeding system on GLRaV-3 viral purifications from both V. vinifera and N. benthamiana and transmits the virus to test plants from plants from both species. Building on this strategy, we used an immunofluorescence approach to localize virions to two retention sites in P. ficus mouthparts. Assays testing molecules capable of blocking virus transmission demonstrated that GLRaV-3-transmission by P. ficus could be disrupted. Our results indicate that our membrane feeding system and transmission-blocking assays are a valid approach and can be used to screen other candidate blocking molecules.

The complete genome sequence of a previously uncharacterized sugarcane‐infecting virus of the genus Ampelovirus was obtained by high‐throughput sequencing (HTS) and rapid amplification of cDNA ends. This genome consists of 13,114 nucleotides (nt) and harbours seven open reading frames. Phylogenetic and pairwise identity analyses with the complete or near‐complete genome sequences of 16 isolates revealed that they all belong to a new Ampelovirus species, family Closteroviridae. These 16 isolates were distributed in three phylogenetic groups and potential recombinant events were identified for only one of these isolates. An immunocapture‐reverse transcription‐PCR (IC‐RT‐PCR) assay was developed with antibodies previously produced against a clostero‐like virus that had not yet been recognized by the International Committee on Taxonomy of Viruses but for which the name sugarcane mild mosaic virus (SCMMV) had been proposed. The new ampelovirus was detected by IC‐RT‐PCR, and Sanger‐sequenced amplicons confirmed the identity of the virus based on HTS data. SCMMV was detected in 65 of 350 samples of quarantined sugarcane germplasm using RT‐PCR targeting the hsp70 gene. These samples originated from Africa, the Caribbean, North America, South America and South‐east Asia, thus revealing that SCMMV is present in many sugarcane‐growing regions of the world. The complete or near‐complete sequences reported here represent the first genomic sequences for SCMMV. Furthermore, the newly developed RT‐PCR diagnostic assay will facilitate the understanding of SCMMV epidemiology and will help improve management practices. [ABSTRACT FROM AUTHOR]

The roles of proteins encoded by members of the genus Ampelovirus, family Closteroviridae are largely inferred by sequence homology or analogy to similarly located ORFs in related viruses. This study employed yeast two-hybrid and bimolecular fluorescence complementation assays to investigate interactions between proteins of grapevine leafroll-associated virus 3 (GLRaV-3). The p5 movement protein, HSP70 homolog, coat protein, and p20B of GLRaV-3 were all found to self-interact, however, the mechanism by which p5 interacts remains unknown due to the absence of a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. Although HSP70h forms part of the virion head of closteroviruses, in GLRaV-3, it interacts with the coat protein that makes up the body of the virion. Silencing suppressor p20B has been shown to interact with HSP70h, as well as the major coat protein and the minor coat protein. The results of this study suggest that the virion assembly of a member of the genus Ampelovirus occurs in a similar but not identical manner to those of other genera in the family Closteroviridae. Identification of interactions of p20B with virus structural proteins provides an avenue for future research to explore the mechanisms behind the suppression of host silencing and suggests possible involvement in other aspects of the viral replication cycle. [ABSTRACT FROM AUTHOR]

This research studied serological relationships and genetic diversity of Argentinean isolates of grapevine leafroll-associated virus-4 (GLRaV-4). Phylogenetic analysis of coat protein (CP) sequences from 19 local isolates revealed clustering with the previously described GLRaV-4 strain 5, strain 6, and strain 9 groups. Evolutionary sequence analysis of the obtained and database-available sequences showed evidence of recombination events. Additionally, both CP N- and C-terminal regions appeared to be under purifying selection, but the N-terminal region presented seven sites under positive selection, with a dN/dS ratio 5-fold greater than that of the C-terminal region. Serological reactivity against monoclonal antibodies supports a higher occurrence probability for linear epitopes in the N-terminal region, as inferred by the sequence analysis. The obtained results reflect an unusual evolutionary behavior of the CP that, together with protein serological reactivity, suggests biological significance of the observed variability. [ABSTRACT FROM AUTHOR]

This article provides an overview of the current epidemiological situation of mealybug wilt of pineapple disease in Ecuador. Mealybug wilt of pineapple (MWP) is a significant virus-associated syndrome that affects pineapple worldwide. The study conducted in Ecuador aimed to determine the prevalence of MWP and the viruses associated with the disease. The results showed a strong association between MWP symptoms and co-infections of PMWaV-2 and PMWaV-1. Other viruses, such as PMWaV-3, PSV-B, PSV-A, PBCOV, and PBERV, were also detected in both symptomatic and symptomless plants. Pineapple mealybugs were present in all sampled plants. Further research is needed to fully understand the etiology of MWP. [Extracted from the article]

Grapevine-infecting ampelo- and vitiviruses are transmitted by scale insects belonging to several species, among which is the European fruit lecanium, Parthenolecanium corni (Bouché) (Hemiptera Coccidae). Our objective was to characterize the transmission biology of grapevine leafroll-associated viruses (GLRaV) and grapevine virus A (GVA) by this soft scale species in order to evaluate its ability to spread these viruses. In transmission experiments with nymphs sampled from different vineyards infected with GLRaV 1, 2, 3 and GVA, P. corni transmitted only GLRaV 1 and GVA to healthy vines. GVA was predominantly transmitted along with GLRaV 1, whereas the latter could be transmitted alone from single or co-infected vines. Vineyard-sampled second instar nymphs were more efficient than first instars at transmitting GLRaV 1, whereas both instars displayed similar transmission rates for GVA. Short virus inoculation access periods and the absence of virus in eggs of females living on infected grapevines fulfilled the criteria of non-circulative semi-persistent transmission mode. [ABSTRACT FROM AUTHOR]

Firespike leafroll-associated virus (FLRaV) is a major pathogen associated with firespike (Odontonema tubaeforme) leafroll disease. Phylogenetic analysis showed that FLRaV possesses typical traits of subgroup II members of ampeloviruses, but encodes two additional proteins, P25 and P37. Here, we determined the microfilament localization of P25 protein. Posttranscriptional gene silencing (PTGS) assay showed that both FLRaV P25 and P37 were able to suppress the local and systemic PTGS and FLRaV P25 was capable of suppressing the green fluorescent protein (GFP) gene silencing triggered by both sense RNA-induced PTGS (S-PTGS) and inverted repeat RNA-induced PTGS (IR-PTGS). In contrast, FLRaV P37 was only able to inhibit the GFP silencing triggered by the S-PTGS but not the IR-PTGS. In the transcriptional gene silencing (TGS) assay, only FLRaV P25 was found to be able to reverse established TGS-mediated silencing of GFP in 16-TGS plants. We also found that FLRaV P25 could aggravate the disease symptom and viral titer of potato virus X in N. benthamiana. These results suggest that FLRaV P25 and P37 may have crucial roles in overcoming host RNA silencing, which provides key insights into our understanding of the molecular mechanisms underlying FLRaV infection. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source–sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease is an increasing problem in all grape-growing regions of the world. The most widespread agent of the disease, Grapevine leafroll-associated virus 3 (GLRaV-3), has never been shown to infect species outside of the genus Vitis. Virus transmission to several plant species used as model systems was tested using the vine mealybug, Planococcus ficus. We show that GLRaV-3 is able to infect Nicotiana benthamiana. Working with GLRaV-3 infected N. benthamiana revealed distinct advantages in comparison with its natural host Vitis vinifera, yielding both higher viral protein and virion concentrations in western blot and transmission electron microscopy observations, respectively. Immunogold labelling of thin sections through N. benthamiana petioles revealed filamentous particles in the phloem cells of GLRaV-3 positive plants. Comparison of assembled whole genomes from GLRaV-3 infected V. vinifera vs. N. benthamiana revealed substitutions in the 5' UTR. These results open new avenues and opportunities for GLRaV-3 research.

Flowering ginger ( Alpinia purpurata ) is economically and culturally important in Hawaii. In the past decade, a slow decline syndrome has impacted the production of this crop in the state. RNA sequencing analyses and virus indexing surveys were done on samples collected from four of the Hawaiian Islands. Viral sequences corresponding to six viruses were recovered from transcriptomic data from samples with virus-like symptoms. Canna yellow mottle virus (CaYMV, genus Badnavirus ) and two novel viruses, Alpinia vein clearing virus (ApVCV, genus Ampelovirus ) and Alpinia vein streaking virus (ApVSV, genus Betanucleorhabdovirus ), were found at a moderate incidence in diseased plants. Conversely, three other viruses, including the two potyviruses, banana bract mosaic virus and bean common mosaic virus, and a badnavirus, banana streak GF virus, were also found but at a low incidence. Virus detection in potential insect vectors and transmission assays identified the mealybug Planococcus citri as a vector of CaYMV and ApVCV, whereas the aphid Pentalonia caladii was identified as a vector of the novel ApVSV. Both P. citri and P. caladii are common pests of flowering ginger in Hawaii. Transmission of ApVSV was achieved using P. caladii colonies either established in the laboratory or naturally feeding on infected plants, although no transmission was obtained using viruliferous aphids originally reared on taro ( Colocasia esculenta ). Our study provides insights into the potential association between viral infections and the observed decline symptoms of flowering ginger in Hawaii. However, more definitive studies are needed to link single or mixed viral infections with decline symptoms., Competing Interests: The author(s) declare no conflict of interest.

Biotic and abiotic stress seriously affects the development of plants, leading to the death of a significant number of plants in natural landscapes. Over the last twelve years, the central zone of Chile has been under an intensive drought, affecting the species inhabiting the Chilean Mediterranean forest, which is classified as a biological hotspot. In this context, our group started a small survey to monitor the presence of intracellular pathogens that may be infecting the plants, increasing the damage caused by the water deprivation. Several plants of one species, Persea lingue, showed symptoms of interveinal yellowing and leaf curling. RNA-seq analyses of one of these samples showed the presence of a long contig with high coverage only in symptomatic plants. Phylogenetic analysis and the molecular features indicated that it was a new species of the Ampelovirus genus. RT-PCR analyses determined the presence of the virus only in symptomatic plants of the same natural preserve.

Grapevine leafroll disease (GLD) is one of the most economically damaging virus diseases in grapevine, with grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) as the main contributors. This study complements a previously published transcriptomic analysis and compared the impact of two different forms of GLD to a symptomless control treatment: a mildly symptomatic form infected with GLRaV-1 and a severe form with exceptionally early leafroll symptoms (up to six weeks before veraison) infected with GLRaV-1 and GLRaV-3. Vine physiology and fruit composition in 17-year-old Pinot noir vines were measured and a gradient of vigor, yield, and berry quality (sugar content and berry weight) was observed between treatments. Virome composition, confirmed by individual RT-PCR, was compared with biological indexing. Three divergent viromes were recovered, containing between four to seven viruses and two viroids. They included the first detection of grapevine asteroid mosaic-associated virus in Switzerland. This virus did not cause obvious symptoms on the indicators used in biological indexing. Moreover, the presence of grapevine virus B (GVB) did not cause the expected corky bark symptoms on the indicators, thus underlining the important limitations of the biological indexing. Transmission of GLRaV-3 alone or in combination with GVB by Planococcus comstocki mealybug did not reproduce the strong symptoms observed on the donor plant infected with a severe form of GLD. This result raises questions about the contribution of each virus to the symptomatology of the plant. [ABSTRACT FROM AUTHOR]

Background: the most widely distributed and virulent Grapevine leafroll-associated viruses (GLRaV) that affect grapevine are GLRaV-1 and GLRaV-3, transmitted semi-persistently by different mealybugs and soft scales, mainly causing downward rolling of the leaf margins and interveinal reddening. Methods: the main objectives of this study were to investigate the genetic structure and molecular diversity of GLRaV-1 and GLRaV-3 in 617 samples from 11 autochthonous Sicilian grapevine cultivars, ascertaining their presence and spread. The detection was implemented by serological and molecular analyses and subsequently phylogenetic analyses on selected Sicilian isolates were conducted. Results: in total, 33 and 138 samples resulted positive to GLRaV-1 and GLRaV-3, with an incidence of 5.34% and 22.36%, respectively; 9 out of the 11 cultivars resulted positive, while the presence of both viruses was not found in 'Grillo' and 'Moscato' cultivars. Conclusions: phylogenetic analyses of the coat protein (CP) gene of 12 GLRaV-1 selected sequences showed a close relationship with European isolates; the discrete nucleotide differentiation and positive selection could demonstrate a current increase in population fitness. The phylogenetic analyses of the CP gene of 31 GLRaV-3 Sicilian CP sequences demonstrates a close relationship between Sicilian and different countries isolates; a certain stability of GLRaV-3 in the different cultivars analyzed is suggested by the discrete differentiation nucleotide and negative selection of the Sicilian isolates. [ABSTRACT FROM AUTHOR]

The roles of proteins encoded by members of the genus Ampelovirus, family Closteroviridae are largely inferred by sequence homology or analogy to similarly located ORFs in related viruses. This study employed yeast two-hybrid and bimolecular fluorescence complementation assays to investigate interactions between proteins of grapevine leafroll-associated virus 3 (GLRaV-3). The p5 movement protein, HSP70 homolog, coat protein, and p20B of GLRaV-3 were all found to self-interact, however, the mechanism by which p5 interacts remains unknown due to the absence of a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. Although HSP70h forms part of the virion head of closteroviruses, in GLRaV-3, it interacts with the coat protein that makes up the body of the virion. Silencing suppressor p20B has been shown to interact with HSP70h, as well as the major coat protein and the minor coat protein. The results of this study suggest that the virion assembly of a member of the genus Ampelovirus occurs in a similar but not identical manner to those of other genera in the family Closteroviridae. Identification of interactions of p20B with virus structural proteins provides an avenue for future research to explore the mechanisms behind the suppression of host silencing and suggests possible involvement in other aspects of the viral replication cycle.

Mealybug wilt of pineapple (MWP) is the most important and complex viral disease affecting pineapple worldwide. High-throughput sequencing was conducted to characterize a new virus identified only in symptomatic pineapple plants and tentatively named pineapple mealybug wilt-associated virus 6 (PMWaV-6). Data analyses revealed a genome of 17,854 nucleotides with an organization resembling members of the genus Ampelovirus, family Closteroviridae. Encoded proteins shared sequence identity with the corresponding proteins of grapevine leafroll-associated virus 3, blackberry vein banding-associated virus, and PMWaV-2. The present study reports the discovery of PMWaV-6, a putative and distinct new member of the genus Ampelovirus, subgroup I, its potential involvement in MWP, and the development of PMWaV-6-specific RT-PCR assays to detect and monitor this virus in field samples. [ABSTRACT FROM AUTHOR]

Abstract 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.

Grapevine-infecting ampelo- and vitiviruses are transmitted by scale insects belonging to several species, among which is the European fruit lecanium, Parthenolecanium corni (Bouché) (Hemiptera Coccidae). Our objective was to characterize the transmission biology of grapevine leafroll-associated viruses (GLRaV) and grapevine virus A (GVA) by this soft scale species in order to evaluate its ability to spread these viruses. In transmission experiments with nymphs sampled from different vineyards infected with GLRaV 1, 2, 3 and GVA, P. corni transmitted only GLRaV 1 and GVA to healthy vines. GVA was predominantly transmitted along with GLRaV 1, whereas the latter could be transmitted alone from single or co-infected vines. Vineyard-sampled second instar nymphs were more efficient than first instars at transmitting GLRaV 1, whereas both instars displayed similar transmission rates for GVA. Short virus inoculation access periods and the absence of virus in eggs of females living on infected grapevines fulfilled the criteria of non-circulative semi-persistent transmission mode.

This article reports on the first identification of Pineapple mealybug wilt-associated virus 1 (PMWaV-1) and 3 (PMWaV-3) in pineapple plants in Malaysia. Mealybug wilt of pineapple (MWP) is a disease that affects pineapple production globally. The study collected leaf samples from farms in Melaka and Johor and used PCR assays to confirm the presence of PMWaV-1, PMWaV-2, and PMWaV-3 in the samples. The findings suggest that PMWaVs could spread through virus-infected pineapple suckers, highlighting the importance of educating and training pineapple farmers in disease prevention and control. [Extracted from the article]

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source–sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development.

Background: the most widely distributed and virulent Grapevine leafroll-associated viruses (GLRaV) that affect grapevine are GLRaV-1 and GLRaV-3, transmitted semi-persistently by different mealybugs and soft scales, mainly causing downward rolling of the leaf margins and interveinal reddening. Methods: the main objectives of this study were to investigate the genetic structure and molecular diversity of GLRaV-1 and GLRaV-3 in 617 samples from 11 autochthonous Sicilian grapevine cultivars, ascertaining their presence and spread. The detection was implemented by serological and molecular analyses and subsequently phylogenetic analyses on selected Sicilian isolates were conducted. Results: in total, 33 and 138 samples resulted positive to GLRaV-1 and GLRaV-3, with an incidence of 5.34% and 22.36%, respectively; 9 out of the 11 cultivars resulted positive, while the presence of both viruses was not found in ‘Grillo’ and ‘Moscato’ cultivars. Conclusions: phylogenetic analyses of the coat protein (CP) gene of 12 GLRaV-1 selected sequences showed a close relationship with European isolates; the discrete nucleotide differentiation and positive selection could demonstrate a current increase in population fitness. The phylogenetic analyses of the CP gene of 31 GLRaV-3 Sicilian CP sequences demonstrates a close relationship between Sicilian and different countries isolates; a certain stability of GLRaV-3 in the different cultivars analyzed is suggested by the discrete differentiation nucleotide and negative selection of the Sicilian isolates.

Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.

Chlorotic fleck disease of ginger, the causal virus of which was unknown so far is an important production constraint of ginger in India and other parts of the world. In the present study, two new RNA viruses were discovered in chlorotic fleck affected plant by the virome analysis using high‐throughput sequencing (HTS) of small RNA and transcriptome. The HTS results were verified through reverse transcriptase polymerase chain reaction (RT‐PCR) using total RNA from infected plants and primers designed from the contigs. Cloning and sequencing of the RT‐PCR products of one of the viruses resulted in a sequence of 4,143 bases that showed similarities to panicoviruses and machlomoviruses. The complete genome of this virus contained six open reading frames (ORFs) that potentially encode proteins of 43, 104, 8, 7, 15 and 27 kDa. Based on the genomic and phylogenetic analysis, this virus is predicted to be a new member of the family Tombusviridae for which the name ginger chlorotic fleck‐associated virus 1 is proposed. Similarly, cloning and sequencing of the RT‐PCR products of the other virus resulted in a sequence of 5,514 bases that showed similarities to ampeloviruses. Based on the genomic and phylogenetic analysis, this virus is predicted to be a new member of the genus Ampelovirus for which the name ginger chlorotic fleck‐associated virus 2 is proposed. Reliable RT‐PCR and SYBR Green‐based quantitative RT‐PCR assays were developed for the detection of both viruses in plants that would aid in the identification and propagation of virus‐free ginger plants. Additional investigations are required to elucidate the relationship between the symptoms and viral infections. [ABSTRACT FROM AUTHOR]

Three novel viruses were detected from Japanese persimmon 'Reigyoku' (Diospyros kaki), which grows poorly when top-grafted onto some interstocks. The genomic characterization and the phylogenetic analysis of these viruses indicated that they are likely new members of the genera Ampelovirus, Polerovirus, and Waikavirus, tentatively named Persimmon ampelovirus (PAmpV), Persimmon polerovirus (PPolV), and Persimmon waikavirus (PWaiV). The alignment of two PAmpV variants with Plum bark necrosis stem pitting–associated virus isolates had 72.5%–79.0% amino acid sequence identities with RNA-dependent RNA polymerase, 71.6%–79.2% with a heat shock protein 70 homolog, and 66.2%–69.8% with coat protein, respectively. Two PPolV variants showed the highest amino acid sequence identities (46.6%) with the P1–P2 fusion protein of Brassica yellows virus. PWaiV had the highest amino acid sequence identity (41.9%) with the conserved domain between proteinase and polymerase of Rice tungro spherical virus. Reverse-transcription and polymerase chain reaction detected Persimmon virus A, Apple crinkle fruit viroid, and Citrus viroid VI from the mother clone with good growth, and PAmpV, PPolV, and Persimmon latent viroid (PLVd) additionally from 20 trees with poor growth. PAmpV, PPolV, and PLVd were detected from four, 10, and 10 of 14 trees with good growth, respectively, and the grafting of budwood harboring these three pathogens caused significant stunt in mother clones. [ABSTRACT FROM AUTHOR]

Cassava is one of the most important staple crops in Africa and its production is seriously damaged by viral diseases. In this study, we identify for the first time and characterize the genome organization of novel ampeloviruses infecting cassava plants in diverse geographical locations using three high-throughput sequencing protocols [Virion-Associated Nucleotide Acid (VANA), dsRNA and total RNA], and we provide a first analysis of the diversity of these agents and of the evolutionary forces acting on them. Thirteen new Closteroviridae isolates were characterized in field-grown cassava plants from the Democratic Republic of Congo (DR Congo), Madagascar, Mayotte, and Reunion islands. The analysis of the sequences of the corresponding contigs (ranging between 10,417 and 13,752 nucleotides in length) revealed seven open reading frames. The replication-associated polyproteins have three expected functional domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). Additional open reading frames code for a small transmembrane protein, a heat-shock protein 70 homolog (HSP70h), a heat shock protein 90 homolog (HSP90h), and a major and a minor coat protein (CP and CPd respectively). Defective genomic variants were also identified in some cassava accessions originating from Madagascar and Reunion. The isolates were found to belong to two species tentatively named Manihot esculenta-associated virus 1 and 2 (MEaV-1 and MEaV-2). Phylogenetic analyses showed that MEaV-1 and MEaV-2 belong to the genus Ampelovirus, in particular to its subgroup II. MEaV-1 was found in all of the countries of study, while MEaV-2 was only detected in Madagascar and Mayotte. Recombination analysis provided evidence of intraspecies recombination occurring between the isolates from Madagascar and Mayotte. No clear association with visual symptoms in the cassava host could be identified.

The etiology of the mealybug wit of pineapple (MWP) disease is associated with a complex of ampeloviruses (Closteroviridae) named Pineapple mealybug wilt-associated virus-1 to -5 (PMWaV-1 to -5). The aims of this study were to determine the specie of PMWaVs associated with MWP symptoms in pineapples plants hybrid 'MD-2', and virus presence in asymptomatic crowns of the same cultivar. Six symptomatic plants and 25 asymptomatic crowns were collected in pineapples cv. 'MD-2' commercial fields in Ciego de Ávila, during the period 2010-2015. The crowns were used as explants to produce "in vitro" plants. Total RNAs were extracted to detect PMWaV-1, -2 and -3 by RT-PCR. Five of the six MWP symptomatic plants contained PMWaV-2 infection, four of them with mixed infection with PMWaV-1 and/or PMWaV-3. The forty percent of the 25 "in vitro" plants were infected, at least, by one PMWaV, 16 % of the three viral species, 8 % by two, and 16 % by only one virus. The incidence of PMWaVs was 38.5 % either for PMWaV-1 and PMWaV-3 (12/31), and 32.3 % for PMWaV-2 (10/31). These results indicate the need of the sanitary certification of pineapple propagation material.

Grapevine leafroll is one of the most widespread and highly destructive grapevine diseases that is responsible for great economic losses to the grape and wine industries throughout the world. Six distinct viruses have been implicated in this disease complex. They belong to three genera, all in the family Closteroviridae. For the sake of convenience, these viruses are named as grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4, -7, and -13). However, their etiological role in the disease has yet to be established. Furthermore, how infections with each GLRaV induce the characteristic disease symptoms remains unresolved. Here, we first provide a brief overview on each of these GLRaVs with a focus on genome structure, expression strategies and gene functions, where available. We then provide a review on the effects of GLRaV infection on the physiology, fruit quality, fruit chemical composition, and gene expression of grapevine based on the limited information so far reported in the literature. We outline key methodologies that have been used to study how GLRaV infections alter gene expression in the grapevine host at the transcriptomic level. Finally, we present a working model as an initial attempt to explain how infections with GLRaVs lead to the characteristic symptoms of grapevine leafroll disease: leaf discoloration and downward rolling. It is our hope that this review will serve as a starting point for grapevine virology and the related research community to tackle this vastly important and yet virtually uncharted territory in virus-host interactions involving woody and perennial fruit crops.

Japanese apricot (Prunus mume Sieb. et Zucc.) is one of the most popular fruit tree species in Japan and is most densely cultivated in Wakayama Prefecture. Since the 1980s, graft-transmissible symptoms, such as leaf-edge necrosis and incomplete flower development, have been observed in Nanko, the major Japanese apricot cultivar in Wakayama. Two ampeloviruses, plum bark necrosis stem pitting-associated virus (PBNSPaV) and little cherry virus 2 (LChV-2) were detected in Nanko trees showing typical symptoms. However, there is currently insufficient epidemiological data to infer that these viruses cause the symptoms. In this study, we conducted a large-scale survey of symptoms and RT-PCR detection of the two viruses in midwestern districts, the major cultivation areas in Wakayama. Among 208 surveyed Nanko trees, two viral infections were significantly associated with incomplete flower development, low fruit bearing rate, and interveinal chlorosis. LChV-2 infection also affected Nanko fruit and stone sizes. Additionally, we showed that the two viruses were already widely distributed throughout the main cultivation area. These results imply that the two ampeloviruses cause overall yield loss in Japanese apricot in Wakayama Prefecture. [ABSTRACT FROM AUTHOR]

Air potato (Dioscorea bulbifera) plants being grown at the Florida Department of Agriculture and Consumer Services Division of Plant Industry Biological Control Laboratory II in Alachua County, Florida were observed exhibiting foliar mosaic symptoms characteristic of virus infection. A double-stranded RNA library generated from a symptomatic plant underwent high-throughput sequencing to determine if viral pathogens were present. Sequence data revealed the presence of two viral genomes, one with properties congruent with members of the genus Potyvirus (family Potyviridae), and the other with members of the genus Ampelovirus (family Closteroviridae). Sequence comparisons and phylogenetic placement indicate that both viruses represent novel species. The names "dioscorea mosaic virus" and "air potato virus 1" are proposed for the potyvirus and ampelovirus, respectively. [ABSTRACT FROM AUTHOR]

Abstract Infectious cDNA clones were developed for Grapevine leafroll-associated virus 3 (GLRaV-3, genus Ampelovirus , family Closteroviridae). In vitro RNA transcripts generated from cDNA clones showed replication via the production of 3′-coterminal subgenomic (sg) mRNAs in Nicotiana benthamiana protoplasts. The detection of sgRNAs and the recovery of progeny recombinant virions from N. benthamiana leaves agroinfiltrated with full-length cDNA clones confirmed RNA replication and virion formation. The 5′ non-translated region (5′ NTR) of GLRaV-3 was exchangeable between genetic variants and complement the corresponding cognate RNA functions in trans. Mutational analysis of the 5′ NTR in minireplicon cDNA clones showed that the conserved 40 nucleotides at the 5′-terminus were indispensable for replication, compared to downstream variable portion of the 5′ NTR. Some of the functional mutations in the 5′ NTR were tolerated in full-length cDNA clones and produced sgRNAs and virions in N. benthamiana leaves, whereas other mutations affected replication and virion formation. [ABSTRACT FROM AUTHOR]

A novel RNA virus was identified in firespike (Odontonema tubaeforme) plants exhibiting leaf curling and chlorosis. The molecular features of the viral genomic RNA and proteins resemble those of ampeloviruses. Based on sequence comparisons and phylogenetic analysis, we propose a new species in the genus Ampelovirus, which we have tentatively named Firespike leafroll-associated virus (FLRaV). Bioassays showed that the virus is mechanically transmissible to Nicotiana benthamiana. In addition, a full-length cDNA clone of FLRaV could successfully infect N. benthamiana via agroinfiltration.

Grapevine leafroll disease is one of the most important virus diseases of grapevines and occurs in every major grape-growing region of the world. The vector-transmission mechanisms of the causative agent, Grapevine leafroll-associated virus 3 (GLRaV-3), remain poorly understood. We show that the vine mealybug, Planococcus ficus, feeds through a membrane feeding system on GLRaV-3 viral purifications from both V. vinifera and N. benthamiana and transmits the virus to test plants from plants from both species. Building on this strategy, we used an immunofluorescence approach to localize virions to two retention sites in P. ficus mouthparts. Assays testing molecules capable of blocking virus transmission demonstrated that GLRaV-3-transmission by P. ficus could be disrupted. Our results indicate that our membrane feeding system and transmission-blocking assays are a valid approach and can be used to screen other candidate blocking molecules.

Grapevine leafroll disease is an increasing problem in all grape-growing regions of the world. The most widespread agent of the disease, Grapevine leafroll-associated virus 3 (GLRaV-3), has never been shown to infect species outside of the genus Vitis . Virus transmission to several plant species used as model systems was tested using the vine mealybug, Planococcus ficus . We show that GLRaV-3 is able to infect Nicotiana benthamiana. Working with GLRaV-3 infected N. benthamiana revealed distinct advantages in comparison with its natural host Vitis vinifera , yielding both higher viral protein and virion concentrations in western blot and transmission electron microscopy observations, respectively. Immunogold labelling of thin sections through N. benthamiana petioles revealed filamentous particles in the phloem cells of GLRaV-3 positive plants. Comparison of assembled whole genomes from GLRaV-3 infected V. vinifera vs. N. benthamiana revealed substitutions in the 5′ UTR. These results open new avenues and opportunities for GLRaV-3 research. [ABSTRACT FROM AUTHOR]

Little cherry disease, caused by little cherry virus 1 (LChV-1) and little cherry virus 2 (LChV-2), which are both members of the family Closteroviridae, severely affects sweet (Prunus avium L.) and sour cherry (P. cerasus L.) orchards lifelong production worldwide. An intensive survey was conducted across different geographic regions of Belgium to study the disease presence on these perennial woody plants and related species. Symptomatic as well as non-symptomatic Prunus spp. trees tested positive via RT-PCR for LChV-1 and -2 in single or mixed infections, with a slightly higher incidence for LChV-1. Both viruses were widespread and highly prevalent in nearly all Prunus production areas as well as in private gardens and urban lane trees. The genetic diversity of Belgian LChV-1 and -2 isolates was assessed by Sanger sequencing of partial genomic regions. A total RNA High-Throughput Sequencing (HTS) approach confirmed the presence of both viruses, and revealed the occurrence of other Prunus-associated viruses, namely cherry virus A (CVA), prune dwarf virus (PDV) and prunus virus F (PrVF). The phylogenetic inference from full-length genomes revealed well-defined evolutionary phylogroups with high genetic variability and diversity for LChV-1 and LChV-2 Belgian isolates, yet with little or no correlation with planting area or cultivated varieties. The global diversity and the prevalence in horticultural areas of LChV-1 and -2 variants, in association with other recently described fruit tree viruses, are of particular concern. Future epidemiological implications as well as new investigation avenues are exhaustively discussed.

Mealybug wilt of pineapple (MWP) is a disease of pineapple that has a long history in Hawaii, but is present throughout the world where pineapples are grown in tropical regions. The disease has an interesting etiology that is poorly understood but involves an association with virus particles, mealybug vectors, and ants which spread the mealybug vectors. Several distinct pineapple mealybug wilt-associated virus (PMWaV) species have been identified thus far with potential further member species yet to be characterized. Pineapple mealybug wilt-associated viruses are member species of the Ampelovirus genus of the Closteroviridae family. Ampeloviruses are split into two subgroups, subgroup I and subgroup II. PMWaV-2 is a subgroup II member, and these have a longer and more complex genome with additional genes on the 3’ terminus of the RNA genome compared to subgroup I ampeloviruses. PMWaV-2, along with the presence of mealybug vectors, have been shown to be necessary factors in symptom development in Hawaii. Some of these extra genes in the 3’ of PMWaV-2 have recently been shown to function as silencing suppressors, and may play a role in the virulence of PMWaV-2 and symptom development. In other regions of the world, reports of symptomatic plants without PMWaV-2 infection, but with PMWaV-1, -3 or some combination, contradict the requirement of PMWaV-2 for symptom development in MWP. It is possible that further, uncharacterized PMWaVs may be present in symptomatic pineapple plants that test negative for PMWaV-2, explaining the inconsistency in symptom development. More research is necessary to explore the confusing etiology of the MWP disease, and to perhaps shed light upon the symptom development.