Grapevine Pinot gris virus spreads in infected vineyards: latent infections have no direct impact on grape production.

BACKGROUND: Grapevine Pinot gris virus (GPGV) infects grapevines worldwide and causes symptoms such as chlorotic mottling and deformations on leaves, stunted shoots and short panicles, or none of these symptoms if it appears as latent infection. So far, the consequences of GPGV infections for winegrowers are difficult to assess since important information such as plant performance at different GPGV infection levels and symptom expression are not fully clarified. METHODS: In order to investigate the course of GPGV spread, annual visual evaluations and ELISA tests were conducted over 3-4 consecutive years in four GPGV-infected vineyards in southern Germany: GEM, HEC, NIM, and REI. The program PATCHY was used to analyze spatial disease patterns. Sanger sequencing was used to determine virus isolates in vines at different GPGV infection levels, to test their respective influence on symptom expression. Yield and GrapeScan (FTIR) analyses were conducted to test the impact of different GPGV infection levels and isolates on fruit quantity and quality. RESULTS: GPGV infections significantly increased in all four vineyards (GEM 22-32%, HEC 50-99%, NIM 83-90%, REI 56-76%) with significant spreading patterns across and along rows. Specific symptom progression patterns were not observed. According to our results, the virus isolate has an influence on whether symptoms develop during a GPGV infection. While yield analyses revealed that yield losses only occur in symptomatic vines and range from 13 to 96% depending on the severity of symptoms, latent infections have no impact on grape production. No relevant effects of GPGV infections on must quality were observed. CONCLUSIONS: Secondary spread of GPGV was observed in all vineyards monitored, indicating vector-borne transmission that is likely to be accelerated by human viticultural management. GPGV should be further monitored to prevent the accumulation of detrimental symptomatic isolates. The results of this study can be used to assess the risk of GPGV to viticulture and should be considered when developing management strategies against the virus.

Whole genome sequence analysis of shallot virus X from India reveals it to be a natural recombinant with positive selection pressure.

BACKGROUND: Shallots are infected by various viruses like Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and Shallot virus X (ShVX). In India, they have been found to be persistently infected by ShVX. ShVX also infects onion and garlic in combination with other carlaviruses and potyviruses. ShVX is a member of genus Allexivirus of family Alphaflexiviridae. ShVX has a monopartite genome, which is represented by positive sense single-stranded RNA. Globally, only six complete and 3 nearly complete genome sequences of ShV X are reported to date. This number is insufficient to measure a taxon's true molecular diversity. Moreover, the complete genome sequence of ShVX from Asia has not been reported as yet. Therefore, this study was undertaken to generate a complete genome sequence of ShVX from India. RESULTS: Shallot virus X (ShVX) is one of the significant threats to Allium crop production. In this study, we report the first complete genome sequence of the ShVX from India through Next-generation sequencing (NGS). The complete genome of the ShVX (Accession No. OK104171), from this study comprised 8911 nucleotides. In-silico analysis of the sequence revealed variability between this isolate and isolates from other countries. The dissimilarities are spread all over the genome specifically some non-coding intergenic regions. Statistical analysis of individual genes for site-specific selection indicates a positive selection in NABP region. The presence of a recombination event was detected in coat protein region. The sequence similarity percentage and phylogenetic analysis indicate ShVX Indian isolate is a distinctly different isolate. Recombination and site-specific selection may have a function in the evolution of this isolate. This is the first detailed study of the ShVX complete genome sequence from Southeast Asia. CONCLUSION: This study presents the first report of the entire genome sequence of an Indian isolate of ShVX along with an in-depth exploration of its evolutionary traits. The findings highlight the Indian variant as a naturally occurring recombinant, emphasizing the substantial role of recombination in the evolution of this viral species. This insight into the molecular diversity of strains within a specific geographical region holds immense significance for comprehending and forecasting potential epidemics. Consequently, the insights garnered from this research hold practical value for shaping ShVX management strategies and providing a foundation for forthcoming studies delving into its evolutionary trajectory.

Exploring the viral landscape of saffron through metatranscriptomic analysis.

Saffron (Crocus sativus L.), a historically significant crop valued for its nutraceutical properties, has been poorly explored from a phytosanitary perspective. This study conducted a thorough examination of viruses affecting saffron samples from Spanish cultivars, using high-throughput sequencing alongside a systematic survey of transcriptomic datasets from Crocus sativus at the Sequence Read Archive. Our analysis unveiled a broad diversity and abundance, identifying 17 viruses across the 52 analyzed libraries, some of which were highly prevalent. This includes known saffron-infecting viruses and previously unreported ones. In addition, we discovered 7 novel viruses from the Alphaflexiviridae, Betaflexiviridae, Potyviridae, Solemoviridae, and Geminiviridae families, with some present in libraries from various locations. These findings indicate that the saffron-associated virome is more complex than previously reported, emphasizing the potential of phytosanitary analysis to enhance saffron productivity.

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.

Genetic Analysis of the Emerging Citrus Yellow Vein Clearing Virus Reveals a Divergent Virus Population in American Isolates.

Citrus yellow vein clearing virus is a previously reported citrus virus from Asia with widespread distribution in China. In 2022, the California Department of Food and Agriculture conducted a multipest citrus survey targeting multiple citrus pathogens including citrus yellow vein clearing virus (CYVCV). In March 2022, a lemon tree with symptoms of vein clearing, chlorosis, and mottling in a private garden in the city of Tulare, California, tested positive for CYVCV, which triggered an intensive survey in the surrounding areas. A total of 3,019 plant samples, including citrus and noncitrus species, were collected and tested for CYVCV using conventional reverse transcription polymerase chain reaction, reverse transcription quantitative polymerase chain reaction, and Sanger sequencing. Five hundred eighty-six citrus trees tested positive for CYVCV, including eight citrus species not previously recorded infected under field conditions. Comparative genomic studies were conducted using 17 complete viral genomes. Sequence analysis revealed two major phylogenetic groups. Known Asian isolates and five California isolates from this study made up the first group, whereas all other CYVCV isolates from California formed a second group, distinct from all worldwide isolates. Overall, the CYVCV population shows rapid expansion and high differentiation indicating a population bottleneck typical of a recent introduction into a new geographic area.

Complete genome sequence of a novel foveavirus isolated from Allium sativum L. in China.

The complete genome sequence of a novel foveavirus identified in garlic (Allium sativum L.) in China was determined using RNA-seq, reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The entire genomic RNA (GenBank accession MT981417) is 8748 nucleotides long excluding the 3'-terminal poly(A) tail and contains five open reading frames (ORFs). These ORFs encode the viral replicase, a triple gene block, and a coat protein. The virus was tentatively named "garlic yellow stripe associated virus" (GarYSaV). Pairwise comparisons of protein sequences show that GarYSaV encodes proteins that share less than 47% identity with those of other foveaviruses, suggesting that it represents a new species in the genus. Phylogenetic analysis of amino acid sequences of the replicase and CP confirm that GarYSaV is a member of the genus Foveavirus. To our knowledge, this is the first report of a foveavirus in a monocot plant.

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing.

Quebec is the third-largest wine grape producing province in Canada, and the industry is constantly expanding. Traditionally, 90% of the grapevine cultivars grown in Quebec were winter hardy and largely dominated by interspecific hybrid Vitis sp. cultivars. Over the years, the winter protection techniques adopted by growers and climate changes have offered an opportunity to establish V. vinifera L. cultivars (e.g., Pinot noir). We characterized the virome of leafroll-infected interspecific hybrid cultivar and compared it to the virome of V. vinifera cultivar to support and facilitate the transition of the industry. A dsRNA sequencing method was used to sequence symptomatic and asymptomatic grapevine leaves of different cultivars. The results suggested a complex virome in terms of composition, abundance, richness, and phylogenetic diversity. Three viruses, grapevine Rupestris stem pitting-associated virus, grapevine leafroll-associated virus (GLRaV) 3 and 2 and hop stunt viroid (HSVd) largely dominated the virome. However, their presence and abundance varied among grapevine cultivars. The symptomless grapevine cultivar Vidal was frequently infected by multiple virus and viroid species and different strains of the same virus, including GLRaV-3 and 2. Our data show that viruses and viroids associated with the highest number of grapevines expressing symptoms included HSVd, GLRaV-3 and GLRaV-2, in gradient order. However, co-occurrence analysis revealed that the presence of GLRaV species was randomly associated with the development of virus-like symptoms. These findings and their implications for grapevine leafroll disease management are discussed.

Date palm virus A: first plant virus found in date palm trees.

In this work, a novel ssRNA (+) viral genomic sequence with gene organization typical of members of the subfamily Quinvirinae (family Betaflexiviridae) was identified using high- throughput sequencing data of date palm obtained from the Sequence Read Archive database. The viral genome sequence consists of 7860 nucleotides and contains five ORFs encoding for the replication protein (Rep), triple gene block proteins 1, 2, 3 (TGB 1, 2, and 3), and coat protein (CP). Phylogenetic analysis based on the Rep and the CP amino acid sequences showed the closest relationship to garlic yellow mosaic-associated virus (GYMaV). Based on the demarcation criteria of the family Betaflexiviridae, this new virus, provisionally named date palm virus A (DPVA), could constitute a member of a novel genus. However, considering that DPVA and GYMaV share the same genomic organization and that they cluster together on the Rep phylogenetic analysis, they could also constitute a novel genus together, highlighting the necessity of a revision of the taxonomic criteria of the family Betaflexiviridae.

The complete genome sequence of a divergent grapevine virus I isolate naturally infecting grapevine in Greece.

A significant number of new members of the genus Vitivirus have been identified recently, mainly due to the advent of high-throughput sequencing (HTS). Grapevine virus I (GVI), which was identified in New Zealand in 2018, is one of these viruses. RNAseq HTS analysis of a Greek grapevine (cv. Daphnia), revealed the presence of a GVI-like isolate (D2-1/19). Sequence analysis confirmed the classification of D2-1/19 as GVI. However, both sequence and phylogenetic data exhibited high levels of variability between D2-1/19 and the previously characterized GVI isolates. This study provides the full-length sequence of a divergent GVI isolate, adding knowledge to the limited information available about this recently identified virus.

A novel foveavirus identified in wild grapevine (Vitis vinifera subsp. sylvestris).

We report the genome sequence of a putative new foveavirus infecting non-cultivated Vitis vinifera, tentatively named "grapevine foveavirus A" (GFVA). This virus was identified by high-throughput sequencing analysis of a European wild Vitis collected in Switzerland. Phylogenetic analysis revealed that this virus clustered with known grapevine virus T (GVT) isolates but was clearly distinct from any of them. If considering the International Committee of Taxonomy of Viruses (ICTV)-suggested foveavirus species demarcation criterion based on sequence similarity in the replicase gene/protein, this virus should be considered a member of a new species closely related to GVT. On the other hand, comparison of capsid gene/protein sequences using the same criteria indicates that GFVA is at the border of species demarcation. Whether this virus represents a highly divergent GVT isolate or a member of a distinct but closely related species is discussed.

Survey and Diversity of Grapevine Pinot gris virus in Algeria and Comprehensive High-Throughput Small RNA Sequencing Analysis of Two Isolates from Vitis vinifera cv. Sabel Revealing High Viral Diversity.

Grapevine Pinot gris virus (GPGV) is a putative causal agent of grapevine leaf mottling and deformation disease that has been reported worldwide throughout the grapevine-growing regions. Fifty-four grapevines collected from five Algerian grapevine-growing regions were tested for the presence of GPGV in phloem tissues. Eight of the tested grapevines were infected by GPGV. Viromes of two selected Vitis vinifera cv. Sabel grapevines infected by GPGV and showing virus-like symptoms were analyzed by small RNA sequencing. Phylogenetic analyses of the partial coding sequence (cds) of the RNA-dependent RNA polymerase (RdRp) domain showed that all Algerian GPGV isolates were grouped with some already-described asymptomatic isolates. This study provides the first survey of the occurrence of GPGV in Algeria. Moreover, Grapevine fleck virus, Grapevine rupestris stem pitting-associated virus, Grapevine virus B, Grapevine rupestris vein feathering virus, Hop stunt viroid and Grapevine yellow speckle viroid 1 were detected in Algeria for the first time.

Identification and molecular characterization of a novel foveavirus from Rubus spp. in Turkey.

A novel plant virus was identified by high-throughput sequencing analysis from a raspberry plant showing slight mottling symptom. The complete genome sequence of this virus is 8645 nucleotides long, including the 5' and 3' UTRs. Its genome contains five ORFs and is very close to members of the genus Foveavirus (Quinvirinae, Betaflexiviridae) in terms of genome organization, TGB presence and the sizes of the RdRp and CP proteins. The novel virus shares 33.5-51.3 % and 23.3-41.3 % nucleotide identity to other genera of the Betaflexifiviridae family based on polymerase (RdRp) and CP genes, respectively. Compared to other foveavirus species, the RdRp protein showed the highest sequence identity (45.3 %) to the RdRp of peach chlorotic mottle virus (PCMV) while the maximal sequence identity for the CP protein was 33.9 % with grapevine rupestris stem pitting-associated virus (GRSPaV). The low nucleotide and amino acid sequence identity with known foveaviruses indicated that it was a novel virus, for which the provisional name "rubus virus 1 (RuV1)" is proposed. The phylogenetic analysis supports the assignment of this virus as a new species of the genus Foveavirus. A survey of 537 Rubus spp. samples grown in six provinces of Turkey, including some symptomatic samples, showed a RuV1 prevalence of 2.2 %, confirming its presence in both raspberry and blackberry plants in a single province, although no obvious association between virus infection and specific symptoms was found.

Complete genome sequence of cherry virus T, a novel cherry-infecting tepovirus.

Double-stranded RNA and total RNA purified from sour cherry leaves (Prunus cerasus, cv. Amarelka Chvalkovicka) was analyzed by high-throughput sequencing. BLAST annotation identified contigs with homology to several already known cherry-infecting viruses (prune dwarf virus, prunus necrotic ringspot virus, prunus virus F, little cherry virus 1) as well as contigs with sequences more distantly related to those of members of the family Betaflexiviridae and in particular to prunus virus T of the genus Tepovirus. The full genome sequence of a putative virus (6,847 nucleotides [nt]; GenBank no. MT090966) was assembled and completed at the genome ends. The genome has a typical tepovirus organization, containing three overlapping open reading frames (ORFs), encoding a replication-associated protein, a movement protein and a capsid protein, respectively. Both its genome organization and its phylogenetic relationships show that the virus belongs to the genus Tepovirus, but considering the species demarcation criteria for the family Betaflexiviridae, it appears to represent a novel virus species, and we propose the name "cherry virus T" (ChVT) for this virus.

Camellia ringspot-associated virus 4, a proposed new foveavirus from Camellia japonica.

One large contig with high sequence similarity to Asian prunus virus 2 was identified by high-throughput sequencing from a camellia (Camellia japonica) tree with ringspot symptoms. The complete genome of this new virus was determined to be 8829 nucleotides long, excluding the 3' poly(A) tail. Its genome organization resembles that of known foveaviruses but contains an additional open reading frame in the 3'-terminal region. Phylogenetic analysis also places this virus with members of the genus Foveavirus in the family Betaflexiviridae in the same subgroup. The virus, which is provisionally named "camellia ringspot-associated virus 4″, shares 50-56% nucleotide sequence identity with other foveaviruses and should represent a new species in the genus.

A complex virome including two distinct emaraviruses associated with virus-like symptoms in Camellia japonica.

Camellia japonica plants manifesting a complex and variable spectrum of viral symptoms like chlorotic ringspots, necrotic rings, yellowing with necrotic rings, yellow mottle, leaves and petals deformations, and flower color-breaking have been studied since 1940, mainly by electron microscopic analyses; however, a strong correlation between the symptoms and one or more well-characterized viruses was never verified. In this work, samples collected from symptomatic plants were analyzed using the next-generation sequencing technique, and a complex virome composed of members of the Betaflexiviridae and Fimoviridae families was identified. In particular, the genomic fragments typical of the emaravirus group were organized in the genomes of two new emaraviruses species, tentatively named Camellia japonica-associated emaravirus 1 and 2. They are the first emaraviruses described in camellia plants and found in symptomatic plants. At the same time, in both symptomatic and asymptomatic plants, five betaflexivirus isolates were detected that, based on amino acid sequence comparisons, can be considered two new isolates of the recently characterized camellia ringspot-associated virus 1 and 2 (CRSaV-1/2). These recently identified betaflexiviruses associated with C. japonica disease show an unusual hyper-conservation of the coat protein at the amino acid level. The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are MN385581, MN532567, MN532565, MN385582, MN532566, MN385573, MN385577, MN385574, MN385578, MN385575, MN385579, MN385576, MN385580, MN557024, MN557025, MN557026, MN557027, and MN557028.

Near-complete genome sequence and biological properties of an allexivirus found in Senna rizzinii in Brazil.

Senna rizzinii is a flowering shrub found mainly in the northeast region of Brazil. Here, we report the coding-complete genome sequence, particle morphology, mode of transmission, and the indicator host responses of an isolate of the putative allexivirus cassia mild mosaic virus (CaMMV) found in S. rizzinii. The virus was transmitted mechanically to Chenopodium amaranticolor, C. quinoa, Gomphrena globosa, which showed local lesions, and S. rizzinii, and S. occidentalis, which were infected systemically. It was also efficiently transmitted to S. rizzinii by grafting. Seed transmission was not observed. The near-complete genome sequence of the virus is 7829 nucleotides in length, containing six open reading frames (ORF), like other allexiviruses.

Application of ZnO Nanorods Based Whispering Gallery Mode Resonator in Optical Immunosensors.

In this research a whispering gallery mode (WGM) resonator based on vertically oriented ZnO nanorods, which were formed on silicon surface (silicon/ZnO-NRs), has been applied in the design of optical immunosensor that was dedicated for the determination of grapevine virus A-type (GVA) proteins. Vertically oriented ZnO-NRs were grown on silicon substrates by atmospheric pressure metal organic chemical vapor deposition (APMOCVD) and the silicon/ZnO-NRs structures formed were characterized by structural and optical methods. Optical characterization demonstrates that silicon/ZnO-NRs-based structures can act as 'whispering gallery mode' (WGM) resonator where quasi-whispering gallery modes (quasi-WGMs) are generated. These quasi-WGMs were experimentally observed in the visible and infrared ranges of the photoluminescence spectra. In order to design an immuno-sensing system the anti-GVA antibodies were immobilized on the surface of silicon/ZnO-NRs and in this way silicon/ZnO-NRs/anti-GVA structure was formed. The immobilization of anti-GVA antibodies and then the interaction of silicon/ZnO-NRs/anti-GVA structure with GVA proteins (GVA-antigens) resulted in an opposite shifts of the WGMs peaks in the visible range of the photoluminescence spectra observed as a defect-related photoluminescence emission of ZnO-NRs. Here designed silicon/ZnO-NRs/anti-GVA immuno-sensing structure demonstrates the sensitivity towards GVA-antigens in the concentration range of 1-200 ng/ml. Bioanalytical applicability of the silicon/ZnO-NRs-based structures in the WGMs registration mode is discussed.

Identification of Pistacia-associated flexivirus 1, a putative mycovirus of the family Gammaflexiviridae, in the mastic tree (Pistacia lentiscus) transcriptome.

In this study, we identified the genome sequence of the novel virus Pistacia-associated flexivirus 1 (PAFV1), a putative member of the mycovirus family Gammaflexiviridae (the order Tymovirales), via analysis of a transcriptome dataset for the mastic tree (Pistacia lentiscus, the family Anacardiaceae). PAFV1 was predicted to have three open reading frames (ORFs): ORF1, encoding a replicase (REP) with RNA-dependent RNA polymerase activity; ORF2, a movement protein (MP); and ORF3, a hypothetical protein. The PAFV1 REP sequence showed high similarity to those of three known members of the family Gammaflexiviridae i.e., Entoleuca gammaflexivirus 1 (EnFV1), Entoleuca gammaflexivirus 2 (EnFV2), and Botrytis virus F (BVF). A genome contig of the fungus Monosporascus cannonballus also contained a sequence of an endogenous virus similar to that of PAFV1. Sequence comparison and phylogenetic analysis indicated that PAFV1, EnFV1, and the endogenous virus of M. cannonballus formed a distinct subgroup (apart from EnFV2 and BVF), and may be the founding members of a novel genus in the family Gammaflexiviridae. Notably, MP sequences of PAFV1/EnFV1 showed similarity to the MP sequences of the mycovirus group called tobamo-like mycoviruses (an unassigned taxon), implying that genomic recombination occurred between members of the family Gammaflexiviridae and tobamo-like mycoviruses. Since PAFV1 is phylogenetically related to mycoviruses, PAFV1 may also be a mycovirus that infected a fungus associated with the mastic tree sample, which is evidenced by the presence of fungal ribosomal RNA sequences in the mastic tree transcriptome. Thus, the PAFV1 genome sequence may be useful in elucidating the genome evolution of Gammaflexiviridae and tobamo-like mycoviruses. Keywords: Pistacia-associated flexivirus 1; Gammaflexiviridae; mycovirus, mastic tree.

Metaviromic analysis reveals coinfection of papaya in western Kenya with a unique strain of Moroccan watermelon mosaic virus and a novel member of the family Alphaflexiviridae.

Severe mottling symptoms were observed on Carica papaya L. in Koyonzo, Kakamega County, Kenya. Total RNA was sequenced via an RNAtag-seq workflow. Assembled contigs indicated the presence of a divergent strain of Moroccan watermelon mosaic virus (genus Potyvirus) with a complete genome length of 9,733 nt (GenBank accession no. MN418119). Additionally, the complete genome sequence of a novel member of the viral genus Allexivirus was determined (GenBank accession no. MN418120). The genome contains six open reading frames (ORFs) that show varying degrees of sequence similarity to members of the genus Allexivirus; however, it appears to lack an ORF encoding a nucleic-acid-binding homolog. The tentative name "papaya virus A" (PaVA) has been proposed for this virus.

Development of RT-qPCR assays for the detection of three latent viruses of pome.

Latent fruit tree viruses present economic threat to the industry and nurseries as diseases they cause not only reduce fruit quality and production yield, but can also be spread inadvertently through propagation due to the lack of viral symptoms on an infected mother plant. As a result, these viruses require appropriate detection tools for effective management. In this study we developed RT-qPCR assays for the detection of three latent viruses of pome, apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), and apple mosaic virus (ApMV), using the alignment of representative sequences from the NCBI database. The optimized assays were shown to be specific by successfully amplifying the target from positive controls without showing any detectable amplification in negative and non-target controls, and revealed high sensitivity by reliably detecting as low as 101 copies per reaction. The results also demonstrated that both the choice of extraction method and the reagents used for RT-qPCRcould play a critical role in virus detection outcome. These assays were both reliable and robust compared to the extant RT-PCR methods, and they could be a viable tool for making informed management decisions.