Identification and molecular characterization of two novel viruses from Allamanda cathartica in China.

Allamanda cathartica is an ornamental medicinal plant that grows widely in the tropics. In the present study, two novel viruses, Allamanda chlorotic virus A (AlCVA) and Allamanda chlorotic virus B (AlCVB), were identified in an A. cathartica plant with interveinal chlorosis by ribosomal RNA-depleted total-RNA sequencing. Phylogenetic analysis and sequence comparisons confirmed that AlCVA and AlCVB belong to the families Closteroviridae and Betaflexiviridae, respectively. Long, flexuous, filamentous virus particles approximately 12 nm in diameter and 784-2291 nm in length were observed using transmission electron microscopy. A specific RT-PCR assay was used to demonstrate a consistent association of viral infection with symptoms.

Influence of mixed and single infection of grapevine leafroll-associated viruses and viral load on berry quality.

Grapevine leafroll disease is a viral disease that affects grapevines (Vitis vinifera L.) and has a severe economic impact on viticulture. In this study, the effect of grapevine leafroll-associated viruses (GLRaV) on berry quality was investigated in clones of cultivar cv. Crimson Seedless table grapes infected with GLRaV. RT-PCR confirmed the identity of the clones: clone 3236, infected only with GLRaV-3 (termed single); clone 3215, infected with GLRaV-3, GLRaV-4 strain 9 and grapevine virus A (termed mixed); and a viral free clone of the same genetic background of the infected clones (termed control). The berry quality indices of size, sugar, acidity and anthocyanin content were measured at harvest maturity. RT-qPCR was used to determine the viral load. The study was repeated over 2 year. A two-way, multivariate analysis of variance was applied with clone and year as independent variables and the measured berry quality parameters as a dependent variable. All dependent variables were significantly affected by viral infection (Wilks, λ, (2,33) = 0.033895, P-value <0.001), while only titratable acidity was affected by year. The average berry dry mass decreased (P-value <0.001). The water content of both infected clones was greater than that of the control (P-value <0.001). Both infected clones displayed reduced sugar content as a fraction of the berry dry mass (P-value <0.001). The anthocyanin and the phenol content of the infected clones were significantly reduced compared with the control clone (P < 0.001, P < 0.05, clone 3236 and clone 3215, respectively). Finally, the viral load was highly variable, and no quantitative relationship between viral load and berry composition was found.

Investigating Protein-Protein Interactions Between Grapevine Leafroll-Associated Virus 3 and Vitis vinifera.

Grapevine leafroll disease (GLD) is a globally important disease that affects the metabolic composition and biomass of grapes, leading to a reduction in grape yield and quality of wine produced. Grapevine leafroll-associated virus 3 (GLRaV-3) is the main causal agent for GLD. This study aimed to identify protein-protein interactions between GLRaV-3 and its host. A yeast two-hybrid (Y2H) library was constructed from Vitis vinifera mRNA and screened against GLRaV-3 open reading frames encoding structural proteins and those potentially involved in systemic spread and silencing of host defense mechanisms. Five interacting protein pairs were identified, three of which were demonstrated in planta. The minor coat protein of GLRaV-3 was shown to interact with 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 02, a protein involved in primary carbohydrate metabolism and the biosynthesis of aromatic amino acids. Interactions were also identified between GLRaV-3 p20A and an 18.1-kDa class I small heat shock protein, as well as MAP3K epsilon protein kinase 1. Both proteins are involved in the response of plants to various stressors, including pathogen infections. Two additional proteins, chlorophyll a-b binding protein CP26 and a SMAX1-LIKE 6 protein, were identified as interacting with p20A in yeast but these interactions could not be demonstrated in planta. The findings of this study advance our understanding of the functions of GLRaV-3-encoded proteins and how the interaction between these proteins and those of V. vinifera could lead to GLD.

Complete genome sequence of a novel closterovirus isolated from Dregea volubilis.

The complete genome sequence of a putative novel closterovirus, tentatively named "Dregea volubilis closterovirus 1" (DvCV1, GenBank accession no. MZ779122), infecting Dregea volubilis in China was determined using high-throughput sequencing (HTS). The complete genome sequence of DvCV1 consists of 16,165 nucleotides (nt) and contains nine ORFs. The genome structure of DvCV1 is typical of members of the genus Closterovirus. Complete genome sequence analysis showed that DvCV1 shares 41.4-48.4% nucleotide sequence identity with other known closteroviruses. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70-like protein (HSP70h), and coat protein (CP) of DvCV1 share 46.80-62.65%, 31.06-51.80%, and 28.34-37.37% amino acid sequence identity, respectively, with the RdRp, HSP70h and CP of other closteroviruses. Phylogenetic analysis based on HSP70h aa sequences placed DvCV1 alongside other members of the genus Closterovirus in the family Closteroviridae. These results suggest that DvCV1 is a new member of the genus Closterovirus. This is the first report of a closterovirus infecting D. volubilis.

Identification of Interactions between Proteins Encoded by Grapevine Leafroll-Associated Virus 3.

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.

Gathered from the Vine: A Survey of Seven Grapevine Viruses Within New England Vineyards.

Vineyards in the Southeastern New England American Viticultural Area were surveyed for the incidence of seven major viruses: grapevine leafroll-associated viruses (GLRaV-1, GLRaV-2, GLRaV-3, and GLRaV-4), grapevine fanleaf virus (GFLV), tomato ringspot virus (ToRSV), and tobacco ringspot virus (TRSV). Viruses were detected by DAS-ELISA and confirmed by RT-PCR and Sanger sequencing. Multiple viruses were present in 19 out of the 25 vineyards surveyed between 2018 and 2020. GLRaV-3 (27.59%) was the most prevalent virus followed by GLRaV-4 (14.90%), GLRaV-1 (13.52%), GLRaV-2 (11.03%), ToRSV (6.34%), GFLV (5.24%), and TRSV (2.62%). Furthermore, phylogenetic analyses of the viral partial genome sequences acquired in this study revealed that the grapevine viruses present in this area are diverse, indicating that they may have been introduced from different sources. Our findings stress the need for improving the sanitary status of planting materials to avoid the introduction and dissemination of viruses to vineyards in this important wine-producing region of New England.

Transcriptomic Analyses of Grapevine Leafroll-Associated Virus 3 Infection in Leaves and Berries of 'Cabernet Franc'.

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.

A novel RNA virus, Thesium chinense closterovirus 1, identified by high-throughput RNA-sequencing of the parasitic plant Thesium chinense.

The genome sequence of a closterovirus (genus Closterovirus, family Closteroviridae), tentatively named Thesium chinense closterovirus 1 (TcCV1), was identified by performing high-throughput RNA-sequencing of the haustoria and root tissues of Thesium chinense, a parasitic plant. The TcCV1 genome was predicted to encode nine proteins, eight of which have orthologs in previously identified closteroviruses. The TcCV1 RNA-dependent RNA polymerase (RdRp) and heat shock protein 70 homolog (Hsp70h) showed 27.8-68.2% and 23.8-55.1% amino acid identity, respectively, to orthologous proteins of known closteroviruses. The putative +1 ribosomal frameshifting site required for producing RdRp was identified as GUUUAGC with UAG stop codon and the skipped nucleotide U. Phylogenetic trees based on RdRp and Hsp70h show that TcCV1 is a novel member of the genus Closterovirus, forming a subclade with a group of known closteroviruses, including mint virus 1 and carnation necrotic fleck virus. The genome sequence of TcCV1 may be useful for studying the genome evolution of closteroviruses. Keywords: Thesium chinense closterovirus 1; Closterovirus; Closteroviridae; Thesium chinense.

Development of a one-step RT-qPCR assay for the detection of Grapevine leafroll-associated virus 7.

Grapevine leafroll disease (GLD) is one of the most economically important viral diseases of grapevines. GLD is caused by a complex of several ssRNA (+) viruses referred to as Grapevine leafroll-associated viruses (GLRaVs). To date, five different GLRaV species have been identified. One of those species, GLRaV-7, was first reported from a symptomless white-fruited wine grape cultivar from Albania. Since its discovery, GLRaV-7 has been reported from 14 countries. Although serological assays have been developed to detect GLRaV-7, commercially available antibodies produce high background signals making them unsuitable for regulatory testing. Furthermore, while molecular detection assays have been shown to be more sensitive when compared to the serological assays, published molecular assays, except the one Reverse Transcription-quantitaive Polymerase Chain Reaction (RT-qPCR) assay based on heat shock protein 70 homologue (HSP70h) gene, have been reported to be inadequate in detecting all reported isolates of GLRaV-7. Availability of multiple assays provides flexibility to diagnostic laboratories in cases where the chosen assay fails to detect a strain or an isolate of a pathogen due to variation in its targeted region or where additional confirmation of the results is required. In this study, we developed a sensitive and specific RT-qPCR assay, based on a region of p61 gene of GLRaV-7, which detected all available isolates.

Transmission of Grapevine Ampelo- and Vitiviruses by the Bohemian Mealybug Heliococcus bohemicus Sulc (Hemiptera: Pseudococcidae).

Grapevine-infecting ampelo- and vitiviruses are transmitted by several scale insect species, including the Bohemian mealybug, Heliococcus bohemicus Sulc. Virus infectivity experiments were performed with this species to study the transmission ability of natural populations living in infected vineyards in Alsace, France. Mealybugs were sampled on vines infected by grapevine leafroll-associated viruses (GLRaV-1, -2, and -3) and by grapevine virus A (GVA), either alone or in combinations. Out of six natural populations tested, only one, located at Bennwihr, was able to transmit GLRaV-1 and -3 to healthy vines, though with low transmission rates (1.6 and 11.8%, respectively). Mealybugs from Bennwihr were also able to transmit GLRaV-3 from grapevines of another location where H. bohemicus was not a vector. Conversely, mealybugs from two other locations did not transmit any virus acquired from infected grapevines at Bennwihr. These results suggest differences in vector ability between H. bohemicus populations. Moreover, laboratory experiments were developed to estimate the minimal acquisition and inoculation access periods (AAP and IAP, respectively) for virus transmission of GLRaV-1 and -3, and GVA. First instar nymphs transmitted GLRaV-1 after 6 h AAP, GLRaV-3 and GVA together after 1 h AAP, and the three viruses after only 1 h IAP, supporting a semi-persistent mode of transmission. Second instar nymphs fed on multi-infected grapevine for 72 h then starved or fed on potatoes tested positive by RT-PCR for GLRaV-1 and -3 after up to 35 and 40 days, respectively, contrasting with the short retention times generally observed for mealybugs. These findings provide new knowledge of the vector ability of H. bohemicus.

Nuances of Responses to Two Sources of Grapevine Leafroll Disease on Pinot Noir Grown in the Field for 17 Years.

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.

Complete genome sequence of peony leafroll-associated virus, a novel ampelovirus in subgroup I.

The complete genome sequence of peony leafroll-associated virus (PLRaV) was determined by deep sequencing of ribosomal-RNA-depleted total RNA extracted from a peony plant exhibiting leafroll symptoms. Further PCR and RACE analysis showed that the PLRaV genome consists of 15,406 nucleotides and contains 10 putative open reading frames, with an organization typical of members of the genus Ampelovirus, family Closteroviridae. Amino acid sequence comparisons showed that the viral heat shock protein 70 homolog (HSP70h) shared the highest sequence identity (41.7%) with the corresponding region of grapevine leafroll-associated virus 1, and the coat protein (CP) and RNA-dependent RNA polymerase (RdRp) shared the highest sequence identity (32.1% and 52.3%, respectively) with grapevine leafroll-associated virus 13. Phylogenetic analysis of the HSP70h, CP, and RdRp aa sequences showed that PLRaV clustered with members of subgroup I of the genus Ampelovirus.

Improved Detection of Little Cherry Virus-2 Using a Hydrolysis Probe to Manage the Pacific Northwest Little Cherry Disease Epidemic.

Little cherry virus-2 (LChV-2) is a viral pathogen that is reaching epidemic levels in Washington State. This virus is insect vectored and has significant impacts on sweet cherry production. To aid growers in making informed management decisions, we sought to develop a diagnostic assay to better detect isolates of LChV-2 currently found in Washington, allowing more accurate estimations of disease occurrence. This study showed that there were two distinct genotypes of LChV-2 present in Washington State. This information was used to develop an up-to-date reverse transcription real-time quantitative PCR assay, which was then optimized, validated, and compared with four previously published assays of a panel of field samples. This comparison demonstrated that the newly developed assay provided greater sensitivity, accurately detecting <10 copies per reaction and could detect both LChV-2 genotypes. Finally, we examined the effect of potential inhibitors in various tissue types from cherry, finding that young leaf tissue affected sensitivity of detection less than root tissues.

Grapevine Leafroll-Associated Virus 3 in Single and Mixed Infections Triggers Changes in the Oxidative Balance of Four Grapevine Varieties.

With the aim to characterize changes caused by grapevine leafroll-associated virus 3 (GLRaV-3) singly or in coinfection with other viruses and to potentially determine genotype-specific or common markers of viral infection, thirty-six parameters, including nutrient status, oxidative stress parameters, and primary metabolism as well as symptoms incidence were investigated in 'Cabernet Franc,' 'Merlot,' 'Pinot Noir,' and 'Tribidrag' grapevine varieties. Host responses were characterized by changes in cellular redox state rather than disturbances in nutrient status and primary metabolic processes. Superoxide dismutase, hydrogen peroxide, and proteins were drastically affected regardless of the type of isolate, the host, and the duration of the infection, so they present cellular markers of viral infection. No clear biological pattern could be ascertained for each of the GLRaV-3 genotypes. There is a need to provide a greater understanding of virus epidemiology in viticulture due to the increasing natural disasters and climate change to provide for global food production security. Finding grape varieties that will be able to cope with those changes can aid in this task. Among the studied grapevine varieties, autochthonous 'Tribidrag' seems to be more tolerant to symptoms development despite numerous physiological changes caused by viruses.

Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties.

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

Transmission of Grapevine leafroll-associated virus-1 (Ampelovirus) and Grapevine virus A (Vitivirus) by the Cottony Grape Scale, Pulvinaria vitis (Hemiptera: Coccidae).

The cottony grape scale Pulvinaria vitis is a scale insect colonizing grapevine; however, its capacity as a vector of grapevine viruses is poorly known in comparison to other scale species that are vectors of viral species in the genera Ampelovirus and Vitivirus. The ability of P. vitis to transmit the ampeloviruses Grapevine leafroll-associated viruses [GLRaV]-1, -3, and -4, and the vitivirus Grapevine virus A (GVA), to healthy vine cuttings was assessed. The scale insects used originated from commercial vine plots located in Alsace, Eastern France. When nymphs sampled from leafroll-infected vineyard plants were transferred onto healthy cuttings, only one event of transmission was obtained. However, when laboratory-reared, non-viruliferous nymphs were allowed to acquire viruses under controlled conditions, both first and second instar nymphs derived from two vineyards were able to transmit GLRaV-1 and GVA. This is the first report of GLRaV-1 and GVA transmission from grapevine to grapevine by this species.

Viromics unveils extraordinary genetic diversity of the family Closteroviridae in wild citrus.

Our knowledge of citrus viruses is largely skewed toward virus pathology in cultivated orchards. Little is known about the virus diversity in wild citrus species. Here, we used a metatranscriptomics approach to characterize the virus diversity in a wild citrus habitat within the proposed center of the origin of citrus plants. We discovered a total of 44 virus isolates that could be classified into species Citrus tristeza virus and putative species citrus associated ampelovirus 1, citrus associated ampelovirus 2, and citrus virus B within the family Closteroviridae, providing important information to explore the factors facilitating outbreaks of citrus viruses and the evolutionary history of the family Closteroviridae. We found that frequent horizontal gene transfer, gene duplication, and alteration of expression strategy have shaped the genome complexity and diversification of the family Closteroviridae. Recombination frequently occurred among distinct Closteroviridae members, thereby facilitating the evolution of Closteroviridae. Given the potential emergence of similar wild-citrus-originated novel viruses as pathogens, the need for surveillance of their pathogenic and epidemiological characteristics is of utmost priority for global citrus production.

TASPERT: Target-Specific Reverse Transcript Pools to Improve HTS Plant Virus Diagnostics.

High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique's success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.

Novel Ampeloviruses Infecting Cassava in Central Africa and the South-West Indian Ocean Islands.

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.