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.

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.

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.

Genotype Sequencing and Phylogenetic Analysis Revealed the Origins of Citrus Yellow Vein Clearing Virus California Isolates.

The Citrus yellow vein clearing virus (CYVCV) causes a viral disease that has been reported in some citrus-growing regions in countries in Eurasia including Pakistan, India, Türkiye, Iran, China, and South Korea. Recently, CYVCV was detected in a localized urban area in a town in the middle of California's citrus-growing region and marks the first occurrence of the virus in North America. CYVCV has been reported to be spread by aphid and whitefly vectors and is graft and mechanically transmitted. Hence, it is an invasive pathogen that presents a significant threat to the California citrus industry, especially lemons, which are highly symptomatic to CYVCV. To elucidate the origin of the CYVCV California strain, we used long-read sequencing technology and obtained the complete genomes of three California CYVCV isolates, CA1, CA2, and CA3. The sequences of these isolates exhibited intergenomic similarities ranging from 95.4% to 97.4% to 54 publicly available CYVCV genome sequences, which indicated a relatively low level of heterogeneity. However, CYVCV CA isolates formed a distinct clade from the other isolates when aligned against other CYVCV genomes and coat protein gene sequences as shown by the neighbor network analysis. Based on the rooted Maximum Likelihood phylogenetic trees, CYVCV CA isolates shared the most recent common ancestor with isolates from India/South Asia. Bayesian evolutionary inferences resulted in a spatiotemporal reconstruction, suggesting that the CYVCV CA lineage diverged from the Indian lineage possibly around 1995. This analysis placed the origin of all CYVCV to around 1990, with South Asia and/or Middle East as the most plausible geographic source, which matches to the first discovery of CYVCV in Pakistan in 1988. Moreover, the spatiotemporal phylogenetic analysis indicated an additional virus diffusion pathway: one from South Asia to China and South Korea. Collectively, our phylogenetic inferences offer insights into the probable dynamics of global CYVCV dissemination, emphasizing the need for citrus industries and regulatory agencies to closely monitor citrus commodities crossing state and international borders.

Viruses of Apple Are Seedborne but Likely Not Vertically Transmitted.

Many viruses occur in apple (Malus domestica (Borkh.)), but no information is available on their seed transmissibility. Here, we report that six viruses infecting apple trees, namely, apple chlorotic leaf spot virus (ACLSV), apple green crinkle-associated virus (AGCaV), apple rubbery wood virus 2 (ARWV2), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), and citrus concave gum-associated virus (CCGaV) occur in seeds extracted from apple fruits produced by infected maternal trees. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR) assays revealed the presence of these six viruses in untreated apple seeds with incidence rates ranging from 20% to 96%. Furthermore, ASPV was detected by RT-PCR in the flesh and peel of fruits produced by infected maternal trees, as well as from seeds extracted from apple fruits sold for fresh consumption. Finally, a large-scale seedling grow-out experiment failed to detect ACLSV, ASGV, or ASPV in over 1000 progeny derived from sodium hypochlorite surface sterilized seeds extracted from fruits produced by infected maternal trees, suggesting no detectable transmission via embryonic tissue. This is the first report on the seedborne nature of apple-infecting viruses.

Complete sequence and genome characterization of miscanthus virus M, a new betaflexivirus from Miscanthus sp.

A novel betaflexivirus, tentatively named "miscanthus virus M" (MiVM), was isolated from Miscanthus sp. The complete genome of MiVM is 7,388 nt in length (excluding the poly(A) tail). It contains five open reading frames and has a genome organization similar to those of members of the families Alphaflexiviridae and Betaflexiviridae (subfamily Quinvirinae). The amino acid sequences of both the replicase and coat protein shared less than 45% identity with the corresponding sequences of members of either family. Phylogenetic analysis confirmed that MiVM belongs to the family Betaflexiviridae and subfamily Quinvirinae but it was too distantly related to be included in any currently recognized genus in this family. We therefore propose that miscanthus virus M represents a new species and a new genus in the family Betaflexiviridae.

Complete genome sequence of a novel robigovirus infecting Mentha arvensis.

The complete genomic sequence of a novel robigovirus, provisionally named "Mentha arvensis robigovirus 1" (MARV1), was determined by combining next-generation sequencing (NGS), reverse transcription polymerase chain reaction (RT-PCR), and rapid amplification of cDNA ends (RACE) PCR. The complete genomic sequence of this new virus is 7617 nucleotides in length, excluding the 3' poly(A) tail. The MARV1 genome encodes a putative replicase, "triple gene block" proteins, and a coat protein. Phylogenetic analysis demonstrated that MARV1 is a member of the genus Robigovirus, with closest relationships to African oil palm ringspot virus (AOPRV). Furthermore, MARV1-derived small interfering RNAs (siRNAs) showed typical patterns of plant-virus-derived siRNAs produced by the host antiviral RNA interference pathway. This is the first report of a plant virus of the genus Robigovirus in M. arvensis.

The functions of triple gene block proteins and coat protein of apple stem pitting virus in viral cell-to-cell movement.

Apple stem pitting virus is a species in the genus Foveavirus in the family Betaflexiviridae. Apple stem pitting virus (ASPV) commonly infects apple and pear plants grown worldwide. In this study, by integrating bimolecular fluorescence complementation, split-ubiquitin-based membrane yeast two-hybrid, and Agrobacterium-mediated expression assays, the interaction relationships and the subcellular locations of ASPV proteins TGBp1-3 and CP in Nicotiana benthamiana leaf cells were determined. Proteins CP, TGBp1, TGBp2, and TGBp3 were self-interactable, and TGBp2 played a role in the formation of perinuclear viroplasm and enhanced the colocalization of TGBp3 with CP and TGBp1. We found that the plant microfilament and endoplasmic reticulum structures were involved in the production of TGBp3 and TGBp2 vesicles, and their disruption decreased the virus accumulation level in the systemic leaves. The TGBp3 motile vesicles functioned in delivering the viral ribonucleoprotein complexes to the plasma membrane. Two cysteine residues at sites 35 and 49 of the TGBp3 sorting signal were necessary for the diffusion of TGBp3-marked vesicles. Furthermore, our results revealed that TGBp1, TGBp2, and CP could increase plasmodesmal permeability and move to the adjacent cells. This study demonstrates an interaction network and a subcellular location map of four ASPV proteins and for the first time provides insight into the functions of these proteins in the movement of a foveavirus.

New Host Plant Species of Grapevine Virus A Identified with Vector-Mediated Infections.

Grapevine virus A (GVA) is an economically important virus and a member of the genus Vitivirus (family Betaflexiviridae) that causes a range of symptoms with qualitative and quantitative effects on grape production. Wild and domesticated species of Vitis, including hybrids used as rootstocks, are considered important natural hosts of GVA. Mechanical transmission to some herbaceous plant species, graft transmission, and vector transmission from grape to grape by various mealybugs and soft scale insects have been reported. Under laboratory and greenhouse conditions, this study demonstrates the transmission of GVA from grapes to alternative hosts by the vine mealybug (Planococcus ficus). Results of ELISA, end-point one-step RT-PCR, and real-time RT-PCR, and in some cases electron microscopy and genome sequencing, confirmed successful transmission to three new plant species commonly found in Croatian vineyards: velvetleaf (Abutilon theophrasti), redroot pigweed (Amaranthus retroflexus), and field poppy (Papaver rhoeas), along with Chenopodium murale and the previously known host Nicotiana benthamiana, with variable infection rates. Depending on the host species, symptoms in the form of leaf reddening, yellow spots, reduced growth of lateral shoots, systemic vein clearing, foliar deformation and rugosity, and dwarfism were observed in GVA-infected plants, whereas no symptoms were observed in infected plants of A. theophrasti. Reverse transmission from these new hosts to grapevines by Pl. ficus was not successful. These results confirm four new GVA host species and open new research venues.

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 Paris polyphylla chlorotic mottle virus infecting Paris polyphylla var. yunnanensis in southwest China.

A novel virus infecting a Paris polyphylla var. yunnanensis plant, tentatively named "Paris polyphylla chlorotic mottle virus" (PpCMV), was discovered in the city of Lijiang, Yunnan Province, China. Its genome consists of 6384 nucleotides (nt), excluding the 3'-terminal poly(A) tail, and contains two open reading frames: ORF1 and ORF2. ORF1 is 6150 nt in length, encoding a large 2050-aa polyprotein with at least two conserved regions encoding a replication-associated protein and a coat protein, the latter of which is located at the 3' end of ORF1. ORF2, consisting of 1185 nt, is located within ORF1 but has a different reading frame. It encodes a 394-aa-long putative movement protein. Phylogenetic analysis based on amino acid sequences revealed that the newly discovered virus exhibited the closest relationship to Hobart betaflexivirus 1 and rhodiola betaflexivirus 1, both of which belong to the genus Capillovirus, sharing 48.8% and 36.5% amino acid sequence identity, respectively, in the structural protein. This is the first report of the complete genome sequence of PpCMV in China.

Identification of divergent isolates of cherry latent virus 1 in Greek sweet cherry orchards.

In this study, samples collected from eight sweet cherry trees in northern Greece were analyzed by high-throughput sequencing for the presence of viruses. Bioinformatic analysis revealed the presence of divergent isolates of cherry latent virus 1 (CLV-1), a recently identified trichovirus in a sweet cherry accession imported into the USA from the Republic of Georgia. The complete genome sequences of seven CLV-1 isolates were determined, and phylogenetic analysis indicated that they belonged to a separate clade from the previously characterized Georgian isolate. A small-scale survey confirmed the presence of CLV-1 in 47 out of 151 sweet cherry samples tested, and partial sequencing of 15 isolates showed a high degree of nucleotide sequence similarity among them.

The genetic variability of grapevine Pinot gris virus (GPGV) in Australia.

Grapevine Pinot gris virus (GPGV; genus Trichovirus in the family Betaflexiviridae) was detected in Australia in 2016, but its impact on the production of nursery material and fruit in Australia is still currently unknown. This study investigated the prevalence and genetic diversity of GPGV in Australia. GPGV was detected by reverse transcription-polymerase chain reaction (RT-PCR) in a range of rootstock, table and wine grape varieties from New South Wales, South Australia, and Victoria, with 473/2171 (21.8%) samples found to be infected. Genomes of 32 Australian GPGV isolates were sequenced and many of the isolates shared high nucleotide homology. Phylogenetic and haplotype analyses demonstrated that there were four distinct clades amongst the 32 Australian GPGV isolates and that there were likely to have been at least five separate introductions of the virus into Australia. Recombination and haplotype analysis indicate the emergence of new GPGV strains after introduction into Australia. When compared with 168 overseas GPGV isolates, the analyses suggest that the most likely origin of Australian GPGV isolates is from Europe. There was no correlation between specific GPGV genotypes and symptoms such as leaf mottling, leaf deformation, and shoot stunting, which were observed in some vineyards, and the virus was frequently found in symptomless grapevines.

Whole genome sequence of Citrus yellow vein clearing virus CA1 isolate.

OBJECTIVES: Citrus yellow vein clearing virus (CYVCV) is an emerging disease that poses a significant threat to the citrus industry in California. In this study, the viral genomic RNA was isolated from Eureka lemon plants in the greenhouse exhibiting CYVCV symptoms. Subsequently, the corresponding DNA genome amplicon was sequenced and annotated. These efforts expand the genotype database of CYVCV, which aims to enhance detection assays, promote understanding of the virus's genetics and evolution, and support the management of this disease. DATA DESCRIPTION: In this report, we present the complete genome sequence of the CYVCV California isolate (CA1). The genome was found to be 7,530 bp in length, with a G + C content of 51.7%. The 5' and 3' termini were determined using 5' and 3' termini rapid amplification of cDNA ends (RACE) systems. Furthermore, our analysis revealed the presence of six open reading frames (ORFs) potentially encoding proteins. All sequence data and annotation have been deposited in GenBank under the accession number OR037276.1.

Molecular characterization of a novel fungal alphaflexivirus reveals potential inter-species horizontal gene transfer.

Sclerotinia sclerotiorum is a notorious phytopathogenic fungus that harbors diverse mycoviruses. A novel positive-sense single-stranded RNA virus, Sclerotinia sclerotiorum alphaflexivirus 2 (SsAFV2), was isolated from the hypovirulent strain 32-9 of S. sclerotiorum, and its complete genome was determined. The SsAFV2 genome contains 7,162 nucleotides (nt), excluding the poly (A) structure, and is composed of four open reading frames (ORF1-4). ORF1 encodes a polyprotein that contains three conserved domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). The ORF3 putative encodes coat proteins (CP), with ORF2 and ORF4 encoding hypothetical proteins of unknown functions. Phylogenetic analysis revealed that SsAFV2 clustered with Botrytis virus X (BVX) based on multiple alignments of helicase, RdRp, and CP, but the methyltransferase of SsAFV2 was most closely related to Sclerotinia sclerotiorum alphaflexivirus 1, suggesting that SsAFV2 is a new member of the Botrexvirus genus within the Alphaflexiviridae family, and also revealed the occurrence of potential inter-species horizontal gene transfer events within the Botrexvirus genus during the evolutionary process. Our results contribute to the current knowledge regarding the evolution and divergence of Botrexviruses.

Molecular diversity of yam virus Y and identification of banana mild mosaic virus isolates infecting yam (Dioscorea spp.).

Two members of the family Betaflexiviridae associated with yam (Dioscorea spp.) have been described so far: yam latent virus (YLV) and yam virus Y (YVY). However, their geographical distribution and molecular diversity remain poorly documented. Using a nested RT-PCR assay, we detected YVY in D. alata, D. bulbifera, D. cayenensis, D. rotundata, and D. trifida in Guadeloupe, and in D. rotundata in Côte d'Ivoire, thus extending the known host range of this virus and geographical distribution. Using amplicon sequencing, we determined that the molecular diversity of YVY in the yam samples analyzed in this work ranged between 0.0 and 29.1% and that this diversity is partially geographically structured. We also identified three isolates of banana mild mosaic virus (BanMMV) infecting D. alata in Guadeloupe, providing the first evidence for BanMMV infection in yam.

Two mutually exclusive evolutionary scenarios for allexiviruses that overcome host RNA silencing and autophagy by regulating viral CRP expression.

The genus Allexivirus currently includes eight virus species that infect allium plants. Previously, we showed that there are two distinct groups of allexiviruses (deletion [D]-type and insertion [I]-type) based on the presence or absence of a 10- to 20-base insert (IS) between the coat protein (CP) and cysteine rich protein (CRP) genes. In the present study of CRPs to analyze their functions, we postulated that evolution of allexiviruses may have been largely directed by CRPs and thus proposed two evolutionary scenarios for allexiviruses based mainly on the presence or absence of IS and determined by how the allexiviruses challenge host resistance mechanisms (RNA silencing and autophagy). We found that both CP and CRP are RNA silencing suppressors (RSS), that they can inhibit each other's RSS activity in the cytoplasm, and that CRP becomes a target of host autophagy in the cytoplasm but not CP. To mitigate CRP interference with CP, and to increase the CP's RSS activity, allexiviruses developed two strategies: confinement of D-type CRP in the nucleus and degradation of I-type CRP by autophagy in the cytoplasm. Here, we demonstrate that viruses of the same genus achieve two completely different evolutionary scenarios by controlling expression and subcellular localization of CRP.

Complete genome sequence of a new virus from Allium sativum L in China.

The complete genome of a new virus belonging to the family Betaflexiviridae was identified in garlic and sequenced by next-generation sequencing and reverse transcription PCR. The complete RNA genome (GenBank accession number OP021693) is 8191 nucleotides in length, excluding the 3' poly(A) tail, and contains five open reading frames (ORFs). These open reading frames encode the viral replicase, triple gene block, and coat protein, and the genome organization is typical of members of the subfamily Quinvirinae. The virus has been tentatively named "garlic yellow curl virus" (GYCV). Phylogenetic analysis suggested that it represents an independent evolutionary lineage in the subfamily, clustering with the currently unclassified garlic yellow mosaic associated virus (GYMaV) and peony betaflexivirus 1 (PeV1). Differences between the phylogenies inferred for the replicase and coat protein indicate that the new virus does not belong to any established genus of the family Betaflexiviridae. This is the first report of GYCV in China.

Evolution and biogeography of apple stem grooving virus.

BACKGROUND: Apple stem grooving virus (ASGV) has a wide host range, notably including apples, pears, prunes and citrus. It is found worldwide. METHOD: In this study, two near complete genomes, and seven coat protein (CP) sequences of Iranian isolates from apple were determined. Sequences added from GenBank provided alignments of 120 genomic sequences (54 of which were recombinant), and 276 coat protein genes (none of them recombinant). RESULT: The non-recombinant genomes gave a well supported phylogeny with isolates from diverse hosts in China forming the base of the phylogeny, and a monophyletic clade of at least seven clusters of isolates from around the world with no host or provenace groupings among them, and all but one including isolates from China. The six regions of the ASGV genome (five in one frame, one - 2 overlapping) gave significantly correlated phylogenies, but individually had less statistical support. The largest cluster of isolates contained those from Iran and had isolates with worldwide provenances, and came from a wide range of mono- and dicotyledonous hosts. Population genetic comparisons of the six regions of the ASGV genome showed that four were under strong negative selection, but two of unknown function were under positive selection. CONCLUSION: ASGV most likely originated and spread in East Asia in one or more of various plant species, but not in Eurasia; the ASGV population of China had the greatest overall nucleotide diversity and largest number of segregating sites.

The interaction between the lemon ribosomal protein ClRPS9-2 and citrus yellow vein clearing virus coat protein affects viral infection and gene silencing suppressor activity.

Citrus yellow vein clearing virus (CYVCV) is an emerging virus that causes serious economic damage to the lemon industry worldwide. The coat protein (CP) of CYVCV is a strong RNA silencing suppressor and is associated with the severity of symptoms in citrus, yet the interaction between CP and host factors remains unknown. In this study, the 40S ribosomal subunit protein S9-2 (ClRPS9-2) was identified as a CP-binding partner using the yeast two-hybrid system from a lemon (cv. Eureka) cDNA library, and the interaction between CP and ClRPS9-2 was demonstrated by in vivo methods. The results suggest that the N-terminal 8-108 amino acid sequence of ClRPS9-2 is crucial for its interaction with CP and may be associated with the nuclear localization of ClRPS9-2. The accumulation and silencing suppressor activity of CP were reduced by transient expression of ClRPS9-2 in Nicotiana benthamiana. Reverse transcription-quantitative PCR analysis showed that the content of CYVCV in ClRPS9-2 transgenic Eureka lemon plants was approximately 50% of that in CYVCV-infected wild-type plants 1 month after inoculation, and mild yellowing and vein clearing symptoms were observed in the transgenic plants. These findings demonstrate that ClRPS9-2 plays a role in host defensive reactions, and the enhanced resistance of transgenic plants to CYVCV may be associated with the up-regulation of salicylic acid-related and R genes.