Spatiotemporal Patterns of Iris Yellow Spot Virus and Its Onion Thrips Vector, Thrips tabaci, in Transplanted and Seeded Onion Fields in New York.

Onion thrips, Thrips tabaci (Lindeman), transmits iris yellow spot virus (IYSV) and is one of the most important pests of Allium crops. IYSV is a member of the species Tospovirus iridimaculaflavi in the genus Orthotospovirus of the family Tospoviridae. This virus typically reduces overall onion bulb quality and weight but can also prematurely kill onion plants. IYSV is neither seed nor mechanically transmitted. Onion fields are typically established via seeds and transplants. A decade ago, onion thrips tended to colonize transplanted fields before seeded fields because plants in transplanted fields were larger and more attractive to thrips than smaller onions in seeded fields. Therefore, we hypothesized that the incidence of IYSV in transplanted fields would be detected early in the season and be spatially aggregated, whereas IYSV would be absent from seeded fields early in the season and initial epidemic patterns would be spatially random. In 2021 and 2022, IYSV incidence and onion thrips populations were quantified in 12 onion fields (four transplanted fields and eight seeded fields) in New York. Fields were scouted four times throughout the growing season (n = 96 samples), and a geospatial and temporal analysis of aggregation and incidence was conducted to determine spatiotemporal patterns in each field type. Results indicated that spatial patterns of IYSV incidence and onion thrips populations were similar early in the season, indicating that transplanted onion fields are no longer the dominant early-season source of IYSV in New York. These findings suggest the need to identify other important early-season sources of IYSV that impact New York onion fields.

Identifying Onion Fields at Risk of Iris Yellow Spot Virus in New York.

Iris yellow spot virus (IYSV) poses a significant threat to dry bulb onion, Allium cepa L., production and can lead to substantial yield reductions. IYSV is transmitted by onion thrips, Thrips tabaci (Lindeman), but not via seed. Transplanted onion fields have been major early season sources of IYSV epidemics. As onion thrips tend to disperse short distances, seeded onion fields bordering transplanted onion fields may be at greater risk of IYSV infection than seeded fields isolated from transplanted ones. Additionally, seeded onion fields planted early may be at greater risk of IYSV infection than those seeded later. In a 2-year study in New York, we compared IYSV incidence and onion thrips populations in seeded onion fields relative to their proximity to transplanted onion fields. In a second study, we compared IYSV incidence in onion fields with either small or large plants during midseason. Results showed similar IYSV incidence and onion thrips populations in seeded onion fields regardless of their proximity to transplanted onion fields, while IYSV incidence was over four times greater in large onion plants than in small ones during midseason. These findings suggest a greater risk of onion thrips-mediated IYSV infection in onion fields with large plants compared with small ones during midseason and that proximity of seeded fields to transplanted ones is a poor indicator of IYSV risk. Our findings on IYSV spread dynamics provided valuable insights for developing integrated pest and disease management strategies for New York onion growers.

Profiling Plant Proteome and Transcriptome Changes during Grapevine Fanleaf Virus Infection.

Viruses can elicit varying types and severities of symptoms during plant host infection. We investigated changes in the proteome and transcriptome of Nicotiana benthamiana plants infected by grapevine fanleaf virus (GFLV) with an emphasis on vein clearing symptom development. Comparative, time-course liquid chromatography tandem mass spectrometry and 3' ribonucleic acid sequencing analyses of plants infected by two wildtype GFLV strains, one symptomatic and one asymptomatic, and their asymptomatic mutant strains carrying a single amino acid change in the RNA-dependent RNA polymerase (RdRP) were conducted to identify host biochemical pathways involved in viral symptom development. During peak vein clearing symptom display at 7 days post-inoculation (dpi), protein and gene ontologies related to immune response, gene regulation, and secondary metabolite production were overrepresented when contrasting wildtype GFLV strain GHu and mutant GHu-1EK802GPol. Prior to the onset of symptom development at 4 dpi and when symptoms faded away at 12 dpi, protein and gene ontologies related to chitinase activity, hypersensitive response, and transcriptional regulation were identified. This systems biology approach highlighted how a single amino acid of a plant viral RdRP mediates changes to the host proteome (∼1%) and transcriptome (∼8.5%) related to transient vein clearing symptoms and the network of pathways involved in the virus-host arms race.

Grapevine Fanleaf Virus RNA1-Encoded Proteins 1A and 1BHel Suppress RNA Silencing.

Grapevine fanleaf virus (GFLV) (genus Nepovirus, family Secoviridae) causes fanleaf degeneration, one of the most damaging viral diseases of grapevines. Despite substantial advances at deciphering GFLV-host interactions, how this virus overcomes the host antiviral pathways of RNA silencing is poorly understood. In this study, we identified viral suppressors of RNA silencing (VSRs) encoded by GFLV, using fluorescence assays, and tested their capacity at modifying host gene expression in transgenic Nicotiana benthamiana expressing the enhanced green fluorescent protein gene (EGFP). Results revealed that GFLV RNA1-encoded protein 1A, for which a function had yet to be assigned, and protein 1BHel, a putative helicase, reverse systemic RNA silencing either individually or as a fused form (1ABHel) predicted as an intermediary product of RNA1 polyprotein proteolytic processing. The GFLV VSRs differentially altered the expression of plant host genes involved in RNA silencing, as shown by reverse transcription-quantitative PCR. In a co-infiltration assay with an EGFP hairpin construct, protein 1A upregulated NbDCL2, NbDCL4, and NbRDR6, and proteins 1BHel and 1A+1BHel upregulated NbDCL2, NbDCL4, NbAGO1, NbAGO2, and NbRDR6, while protein 1ABHel upregulated NbAGO1 and NbRDR6. In a reversal of systemic silencing assay, protein 1A upregulated NbDCL2 and NbAGO2 and protein 1ABHel upregulated NbDCL2, NbDCL4, and NbAGO1. This is the first report of VSRs encoded by a nepovirus RNA1 and of two VSRs that act either individually or as a predicted fused form to counteract the systemic antiviral host defense, suggesting that GFLV might devise a unique counterdefense strategy to interfere with various steps of the plant antiviral RNA silencing pathways during infection. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

ICTV Virus Taxonomy Profile: Secoviridae 2022.

Members of the family Secoviridae are non-enveloped plant viruses with mono- or bipartite linear positive-sense ssRNA genomes with a combined genome of 9 to 13.7 kb and icosahedral particles 25-30 nm in diameter. They are related to picornaviruses and are members of the order Picornavirales. Genera in the family are distinguished by the host range, vector, genomic features and phylogeny of the member viruses. Most members infect dicotyledonous plants, and many cause serious disease epidemics. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Secoviridae, which is available at ictv.global/report/secoviridae.

Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2.

BACKGROUND: The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost 1 million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. RESULTS: We present the 'Mini-XT' miniaturized tagmentation-based library preparation protocol available on protocols.io ( https://doi.org/10.17504/protocols.io.bvntn5en ). SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. The final version of Mini-XT has been used to sequence 4384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. CONCLUSIONS: The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.

Identification of protein interactions of grapevine fanleaf virus RNA-dependent RNA polymerase during infection of Nicotiana benthamiana by affinity purification and tandem mass spectrometry.

The RNA-dependent RNA polymerase (1EPol) is involved in replication of grapevine fanleaf virus (GFLV, Nepovirus, Secoviridae) and causes vein clearing symptoms in Nicotiana benthamiana. Information on protein 1EPol interaction with other viral and host proteins is scarce. To study protein 1EPol biology, three GFLV infectious clones, i.e. GHu (a symptomatic wild-type strain), GHu-1EK802G (an asymptomatic GHu mutant) and F13 (an asymptomatic wild-type strain), were engineered with protein 1EPol fused to a V5 epitope tag at the C-terminus. Following Agrobacterium tumefaciens-mediated delivery of GFLV clones in N. benthamiana and protein extraction at seven dpi, when optimal 1EPol:V5 accumulation was detected, two viral and six plant putative interaction partners of V5-tagged protein 1EPol were identified for the three GFLV clones by affinity purification and tandem mass spectrometry. This study provides insights into the protein interactome of 1EPol during GFLV systemic infection in N. benthamiana and lays the foundation for validation work.

Identification and characterization of a novel virus associated with an eriophyid mite in extracts of fruit trees leaves.

Eriophyid mites are commonly found on the leaf surface of different plant species. In the present study, a novel virus associated with an eriophyid mite species was detected using high-throughput sequencing (HTS) of total RNA from fruit tree leaves, primarily growing under greenhouse conditions. The complete genome sequence was characterized using rapid amplification of cDNA ends followed by Sanger sequencing, revealing a genome of 8885 nucleotides in length. The single positive-stranded RNA genome was predicted to encode typical conserved domains of members of the genus Iflavirus in the family Iflaviridae. Phylogenetic analysis showed this virus to be closely related to the unclassified iflavirus tomato matilda associated virus (TMaV), with a maximum amino acid sequence identity of 59% in the RNA-dependent RNA polymerase domain. This low identity value justifies the recognition of the novel virus as a potential novel iflavirus. In addition to a lack of graft-transmissibility evidence, RT-PCR and HTS detection of this virus in the putative host plants were not consistent through different years and growing seasons, raising the possibility that rather than a plant virus, this was a virus infecting an organism associated with fruit tree leaves. Identification of Tetra pinnatifidae HTS-derived contigs in all fruit tree samples carrying the novel virus suggested this mite as the most likely host of the new virus (p-value < 1e-11), which is tentatively named "eriophyid mite-associated virus" (EMaV). This study highlights the importance of a careful biological study before assigning a new virus to a particular plant host when using metagenomics data.

Grapevine Red Blotch Virus Is Transmitted by the Three-Cornered Alfalfa Hopper in a Circulative, Nonpropagative Mode with Unique Attributes.

The transmission mode of grapevine red blotch virus (GRBV, genus Grablovirus, family Geminiviridae) by Spissistilus festinus, the three-cornered alfalfa hopper, is unknown. By analogy with other members in the family Geminiviridae, we hypothesized circulative, nonpropagative transmission. Time-course experiments revealed GRBV in dissected guts, hemolymph, and heads with salivary glands after a 5-, 8-, and 10-day exposure to infected grapevines, respectively. After a 15-day acquisition on infected grapevines and subsequent transfer on alfalfa, a nonhost of GRBV, the virus titer decreased over time in adult insects, as shown by quantitative PCR. Snap bean proved to be a feeding host of S. festinus and a pseudosystemic host of GRBV after Agrobacterium tumefaciens-mediated delivery of an infectious clone. The virus was efficiently transmitted by S. festinus from infected snap bean plants to excised snap bean trifoliates (90%) or grapevine leaves (100%) but less efficiently from infected grapevine plants to excised grapevine leaves (10%) or snap bean trifoliates (67%). Transmission of GRBV also occurred trans-stadially but not via seeds. The virus titer was significantly higher in (i) guts and hemolymph relative to heads with salivary glands, and (ii) adults emanating from third compared with first instars that emerged on infected grapevine plants and developed on snap bean trifoliates. This study demonstrated circulative, nonpropagative transmission of GRBV by S. festinus with an extended acquisition access period compared with other viruses in the family Geminiviridae and marked differences in transmission efficiency between grapevine, the natural host, and snap bean, an alternative herbaceous host.

ICTV Virus Taxonomy Profile: Closteroviridae.

Viruses in the family Closteroviridae have a mono-, bi- or tripartite positive-sense RNA genome of 13-19 kb, and non-enveloped, filamentous particles 650-2200 nm long and 12 nm in diameter. They infect plants, mainly dicots, many of which are fruit crops. This is a summary of the ICTV Report on the family Closteroviridae, which is available at ictv.global/report/closteroviridae.

Single-cell transcriptomic profiling provides insights into retinal endothelial barrier properties.

Purpose: To better characterize retinal endothelial barrier properties through analysis of individual transcriptomes of primary bovine retinal microvascular endothelial cells (RMECs). Methods: Individual RMECs were captured on the Fluidigm C1 system, cDNA libraries were prepared using a Nextera XT kit, and sequencing was performed on a NextSeq system (Illumina). Data analysis was performed using R packages Scater, SC3, and Seurat, and the browser application Automated Single-cell Analysis Pipeline (ASAP). Alternative splicing events in single cells were quantified with Outrigger. Cytoscape was used for network analyses. Results: Application of a single-cell RNA sequencing (scRNA-seq) analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main differences related to proliferation status. Expression of markers from along the arteriovenous tree suggested that most cells originated from capillaries. Average gene expression levels across all cells were used to develop an in silico model of the inner blood-retina barrier incorporating junctional proteins not previously reported within the retinal vasculature. Correlation of barrier gene expression among individual cells revealed a subgroup of genes highly correlated with PECAM-1 at the center of the correlation network. Numerous alternative splicing events involving exons within microvascular barrier genes were observed, and in many cases, individual cells expressed one isoform exclusively. Conclusions: We optimized a workflow for single-cell transcriptomics in primary RMECs. The results provide fundamental insights into the genes involved in formation of the retinal-microvascular barrier.

Proposed revision of the family Secoviridae taxonomy to create three subgenera, "Satsumavirus", "Stramovirus" and "Cholivirus", in the genus Sadwavirus.

We present a taxonomic proposal for revision of the family Secoviridae, a taxon of plant viruses in the order Picornavirales. We propose the reorganization of the genus Sadwavirus to create three new subgenera and to update the classification of five existing species. The proposed subgenera are "Satsumavirus" (one species: Satsuma dwarf virus), "Stramovirus" (two species: Strawberry mottle virus and Black raspberry necrosis virus) and "Cholivirus" (two species: Chocolate lily virus A and Dioscorea mosaic associated virus).

The Identity of a Single Residue of the RNA-Dependent RNA Polymerase of Grapevine Fanleaf Virus Modulates Vein Clearing in Nicotiana benthamiana.

The mechanisms underlying host plant symptom development upon infection by viruses of the genus Nepovirus in the family Secoviridae, including grapevine fanleaf virus (GFLV), are poorly understood. In the systemic host Nicotiana benthamiana, GFLV strain GHu produces characteristic symptoms of vein clearing in apical leaves, unlike other GFLV strains such as F13, which cause an asymptomatic infection. In this study, we expanded on earlier findings and used reverse genetics to identify residue 802 (lysine, K) of the GFLV-GHu RNA1-encoded RNA-dependent RNA polymerase (1EPol) as a modulator of vein-clearing symptom development in N. benthamiana. Mutations to this site abolished (K to G, A, or Q) or attenuated (K to N or P) symptom expression. Noteworthy, residue 802 is necessary but not sufficient for vein clearing, as GFLV-F13 RNA1 carrying K802 remained asymptomatic in N. benthamiana. No correlation was found between symptom expression and RNA1 accumulation, as shown by reverse transcription-quantitative polymerase chain reaction. Additionally, the involvement of RNA silencing of vein clearing was ruled out by virus-induced gene silencing experiments and structure predictions for protein 1EPol suggested that residue 802 is flanked by strongly predicted stable secondary structures, including a conserved motif of unknown function (805LLKT/AHLK/RT/ALR814). Together, these results reveal the protein nature of the GFLV-GHu symptom determinant in N. benthamiana and provide a solid basis for probing and determining the virus-host proteome network for symptoms of vein clearing.

Iris Yellow Spot Virus Prolongs the Adult Lifespan of Its Primary Vector, Onion Thrips (Thrips tabaci) (Thysanoptera: Thripidae).

Iris yellow spot virus (IYSV) from the genus Tospovirus, family Peribunyaviridae, reduces yield in several crops, especially Allium spp. IYSV is primarily transmitted by onion thrips (Thrips tabaci), but little is known about how IYSV impacts the biology of its principal vector. In a controlled experiment, the effect of IYSV on the lifespan and fecundity of onion thrips was examined. Larvae were reared on IYSV-infected onions until pupation. Individual pupae were confined until adults eclosed, and the lifespan and total progeny produced per adult were monitored daily. Thrips were tested for the virus in reverse-transcriptase polymerase chain reaction using specific primers to confirm the presence of IYSV. Results indicated that 114 and 35 out of 149 eclosing adults tested positive (viruliferous) and negative (nonviruliferous) for IYSV, respectively. The viruliferous adults lived 1.1-6.1 d longer (average of 3.6 d) than nonviruliferous adults. Fecundity of viruliferous and nonviruliferous onion thrips was similar with 2.0 ± 0.1 and 2.3 ± 0.3 offspring produced per female per day, respectively. Fecundity for both viruliferous and nonviruliferous thrips also was significantly positively correlated with lifespan. These findings suggest that the longer lifespan of viruliferous onion thrips adults may allow this primary vector of IYSV to infect more plants, thereby exacerbating IYSV epidemics.

A novel grablovirus from non-cultivated grapevine (Vitis sp.) in North America.

During screening of non-cultivated (wild) grapevine (Vitis sp.) from Napa County, California for the grapevine red blotch virus (GRBV; genus Glabrovirus, family Geminiviridae), an atypical polymerase chain reaction product pattern was observed. Rolling circle amplification followed by cloning and sequencing revealed the presence of a circular DNA characteristic of geminiviruses. The complete genome of nine isolates of the virus ranged from 3204 to 3278 nt in size. The genome most closely resembled that of GRBV in both sequence (57 to 59% identity) and organization. With limited sequence identity to described geminiviruses, this virus warrants designation as a new species, and the name 'Wild Vitis virus 1' is proposed.

Insights Into the Ecology of Grapevine red blotch virus in a Diseased Vineyard.

Limited information is available on the spread of Grapevine red blotch virus (GRBV, genus Grablovirus, family Geminiviridae) in vineyards. To investigate ecological aspects of red blotch disease spread, sticky cards to catch flying insects were placed in 2015 (April to November) and 2016 (March to November) in a vineyard study site in California where disease incidence increased by nearly 20% between 2014 and 2016. Subsets of insect species or taxa were removed from sticky card traps and individual specimens were tested for the presence of GRBV by multiplex polymerase chain reaction. GRBV was consistently detected in Spissistilus festinus (Membracidae), Colladonus reductus (Cicadellidae), Osbornellus borealis (Cicadellidae), and a Melanoliarus sp. (Cixiidae). Populations of these four candidate vectors peaked from June to September, with viruliferous S. festinus peaking from late June to early July in both years. An assessment of co-occurrence and covariation between the spatial distribution of GRBV-infected vines and viruliferous insects identified a significant association only with viruliferous S. festinus. These findings revealed the epidemiological relevance of S. festinus as a vector of GRBV in a vineyard ecosystem. Sequencing coat protein and replicase-associated protein gene fragments of GRBV isolates from newly infected vines and viruliferous vector candidates further suggested secondary spread primarily from local sources and occasionally from background sources.

Causative Role of Grapevine Red Blotch Virus in Red Blotch Disease.

Grapevine red blotch virus (GRBV) has a monopartite single-stranded DNA genome and is the type species of the genus Grablovirus in the family Geminiviridae. To address the etiological role of GRBV in the recently recognized red blotch disease of grapevine, infectious GRBV clones were engineered from the genome of each of the two previously identified phylogenetic clades for Agrobacterium tumefaciens-mediated inoculations of tissue culture-grown Vitis spp. plants. Following agroinoculation and one or two dormancy cycles, systemic GRBV infection was detected by multiplex polymerase chain reaction (PCR) in Vitis vinifera exhibiting foliar disease symptoms but not in asymptomatic vines. Infected rootstock genotype SO4 (V. berlandieri × V. riparia) exhibited leaf chlorosis and cupping, while infection was asymptomatic in agroinoculated 110R (V. berlandieri × V. rupestris), 3309C (V. riparia × V. rupestris), and V. rupestris. Spliced GRBV transcripts of the replicase-associated protein coding region accumulated in leaves of agroinfected vines, as shown by reverse-transcription PCR; this was consistent with systemic infection resulting from virus replication. Additionally, a virus progeny identical in nucleotide sequence to the infectious GRBV clones was recovered from agroinfected vines by rolling circle amplification, cloning, and sequencing. Concomitantly, subjecting naturally infected grapevines to microshoot tip culture resulted in an asymptomatic plant progeny that tested negative for GRBV in multiplex PCR. Altogether, our agroinoculation and therapeutic experiments fulfilled Koch's postulates and revealed the causative role of GRBV in red blotch disease.

The Distribution and Detection of Grapevine red blotch virus in its Host Depend on Time of Sampling and Tissue Type.

Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative polymerase chain reaction (qPCR) method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a pattern similar to that found for the greenhouse-grown plants but, most strikingly, close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.

Importance of Transplanted Onions Contributing to Late-Season Iris yellow spot virus Epidemics in New York.

Iris yellow spot virus (IYSV) is an economically significant tospovirus of onion transmitted by onion thrips (Thrips tabaci Lindeman). IYSV epidemics in onion fields are common in New York; however, the role of various habitats contributing to viruliferous onion thrips populations and IYSV epidemics is not known. In a 2-year field study in New York, the abundance of dispersing onion thrips, including those determined to be viruliferous via reverse-transcriptase polymerase chain reaction, was recorded in habitats known to harbor both IYSV and its vector. Results showed that viruliferous thrips were encountered in all habitats; however, transplanted onion sites accounted for 49 to 51% of the total estimated numbers of viruliferous thrips. During early to midseason, transplanted onion sites had 9 to 11 times more viruliferous thrips than the other habitats. These results indicate that transplanted onion fields are the most important habitat for generating IYSV epidemics in all onion fields (transplanted and direct-seeded) in New York. Our findings suggest that onion growers should control onion thrips in transplanted fields early in the season to minimize risk of IYSV epidemics later in the season.