Acquisition and transmission of Grapevine fanleaf virus (GFLV) by Xiphinema index and Xiphinema italiae (Longidoridae).

Grapevine fanleaf virus (GFLV) is one of the most severe virus diseases of grapevines, causing fanleaf degeneration that is transmitted by Xiphinema index. This paper aims to isolate Xiphinema species from Tunisian vineyard soil samples and assess their ability to acquire and transmit GFLV under natural and controlled conditions. Based on morphological and morphometric analyses, Tunisian dagger nematodes were identified as X. index and Xiphinema italiae. These results were confirmed with molecular identification tools using species-specific polymerase chain reaction primers. The total RNA of GFLV was extracted from specimens of Xiphinema and amplified based on real-time polymerase chain reaction using virus-specific primers. Our results showed that X. index could acquire and transmit the viral particles of GFLV. This nepovirus was not detected in X. italiae, under natural conditions; however, under controlled conditions, this nematode was able to successfully acquire and transmit the viral particles of GFLV.

The complete genome sequence of tomato necrotic ringspot virus in chilli in Thailand derived from next-generation sequencing.

Tomato necrotic ringspot virus (TNRV) was first reported in Thailand in 2011, where it continues to reduce the yield and quality of pepper and tomato crops. Here, we report the complete genome sequence of TNRV isolate chilli-CR derived from next-generation sequencing. The TNRV genome comprises 16,595 nucleotides (nt) on three RNA segments. The L RNA is 8,858 nt, the M RNA is 4,724 nt, and the S RNA is 3,013 nt in length. The genome structure and organization are typical of orthotospoviruses, encoding five proteins, named L, NSm, GNGC, NSs, and N. Pairwise comparison of each genomic RNA segment and its deduced amino acid (aa) sequence showed that TNRV chilli-CR shares 73.6-82.3% nt sequence identity and 81.1-91.9% aa sequence identity with pepper chlorotic spot virus (PCSV). Similar phylogenetic groupings were observed based on each genomic RNA or deduced aa sequence, and with concatenated genomic RNA sequences. The clustering of TNRV and PCSV in all phylogenetic analyses, and the 78.9% overall nt sequence identity observed using the concatenated genomic RNAs suggest that TNRV is a distinct orthotospovirus and that analysis of concatenated orthotospovirus genome sequences will be of value in future phylogenetic studies of this virus group.

Development of Stable Infectious cDNA Clones of Tomato Black Ring Virus Tagged with Green Fluorescent Protein.

Tomato black ring virus (TBRV) is a member of the Nepovirus genus in the Secoviridae family, which infects a wide range of important crop species worldwide. In this work, we constructed four cDNA infectious clones of the TBRV tagged with the green fluorescent protein (TBRV-GFP), which varied in (i) the length of the sequences flanking the GFP insert, (ii) the position of the GFP insert within the RNA2 polyprotein, and (iii) the addition of a self-cutting 2A protein. The presence of the GFP coding sequence in infected plants was verified by RT-PCR, while the infectivity and stability of the constructs were verified by mechanical inoculation of the host plants. The systemic spread of TBRV-GFP within plants was observed under UV light at a macroscopic level, monitoring GFP-derived fluorescence in leaves, and at a microscopic level using confocal microscopy. The obtained clones are a valuable tool for future studies of TBRV-host interactions, virus biology, and the long-term monitoring of its distribution in infected plants.

AlphaFold modeling of nepovirus 3C-like proteinases provides new insights into their diverse substrate specificities.

The majority of picornaviral 3C proteinases (3Cpro) cleavage sites possess glutamine at the P1 position. Plant nepovirus 3C-like proteinases (3CLpro) show however much broader specificity, cleaving not only after glutamine, but also after several basic and hydrophobic residues. To investigate this difference, we employed AlphaFold to generate structural models of twelve selected 3CLpro, representing six substrate specificities. Generally, we observed favorable correlations between the architecture and charge of nepovirus proteinase S1 subsites and their ability to accept or restrict larger residues. The models identified a conserved aspartate residue close to the P1 residue in the S1 subsites of all nepovirus proteinases examined, consistent with the observed strong bias against negatively-charged residues at the P1 position of nepovirus cleavage sites. Finally, a cramped S4 subsite along with the presence of two unique histidine and serine residues explains the strict requirement of the grapevine fanleaf virus proteinase for serine at the P4 position.

Global Genetic Diversity of Tobacco Ringspot Virus Including Newly Reported Isolates from Cotton (Gossypium hirsutum) in Oklahoma.

Cotton is one of the most salient cash crops globally and in the United States. Lately, several virus-like diseases have been reported from cotton in the United States such as the tobacco ringspot virus (TRSV) in Oklahoma. TRSV has been reported from various hosts worldwide with minimal phylogenetic examination. In this study, complete genome sequences of four TRSV isolates from cotton were isolated, and the genetic diversity was investigated along with additional available TRSV isolates retrieved from GenBank. Phylogenetic analysis based on the complete RNA1 and RNA2 sequences distributed all TRSV isolates into three major phylogenetic clades exhibiting a differential clade composition depending on the segment. The TRSV cotton isolates exhibited differential grouping between the RNA1 and RNA2 analyses. Additionally, monophyletic subclades of isolates appeared to be conserved between both segments. Thirty-five recombination events in RNA1 and 23 in RNA2 were identified with implications in the variation of the phylogenetic analyses. Furthermore, multiple hypotheses of TRSV evolution were generated based on the phylogenetic analyses, but to test them, more complete genomes of TRSV will be needed. This study provides the first complete genome analysis of TRSV isolates infecting cotton in the United States and a detailed analysis of global TRSV isolates.

Identification, Sequencing, and Molecular Analysis of RNA2 of Artichoke Italian Latent Virus Isolates from Known Hosts and a New Host Plant Species.

Despite its first description in 1977 and numerous reports of its presence in various plant species in many countries, the molecular information available in GenBank for artichoke Italian latent virus (AILV) is still limited to a single complete genome sequence (RNA1 and 2) of a grapevine isolate (AILV-V) and a partial portion of the RNA2 sequence from an isolate of unknown origin and host. Here, we report the results of molecular analyses conducted on the RNA2 of some AILV isolates, sequenced for the first time in this study, together with the first-time identification of AILV in a new host plant species, namely chard (Beta vulgaris subsp. vulgaris), associated with vein clearing and mottling symptoms on leaves. The different AILV isolates sequenced were from artichoke (AILV-C), gladiolus (AILV-G), Sonchus (AILV-S), and chard (AILV-B). At the molecular level, the sequencing results of the RNA2 segments showed that AILV-C, AILV-G, AILV-S, and AILV-B had a length of 4629 nt (excluding the 3' terminal polyA tail), which is one nt shorter than that of the AILV-V reported in GenBank. A comparison of the RNA2 coding region sequences of all the isolates showed that AILV-V was the most divergent isolate, with the lowest sequence identities of 83.2% at the nucleotide level and 84.7% at the amino acid level. Putative intra-species sequence recombination sites were predicted among the AILV isolates, mainly involving the genomes of AILV-V, AILV-C, and AILV-B. This study adds insights into the variability of AILV and the occurrence of recombination that may condition plant infection.

Betula pendula trees infected by birch idaeovirus and cherry leaf roll virus: Impacts of urbanisation and NO2 levels.

Viruses are frequently a microbial biocontaminant of healthy plants. The occurrence of the infection can be also due to environmental stress, like urbanisation, air pollution and increased air temperature, especially under the ongoing climate change. The aim of the present study was to investigate the hypothesis that worsened air quality and fewer green areas may favour the higher frequency of common viral infections, particularly in a common tree in temperate and continental climates, Betula pendula ROTH. We examined 18 trees, during the years 2015-2017, the same always for each year, in the region of Augsburg, Germany. By specific PCR, the frequency of two viruses, Cherry leaf roll virus (CLRV, genus Nepovirus, family Secoviridae), which is frequent in birch trees, and a novel virus tentatively named birch idaeovirus (BIV), which has been only recently described, were determined in pollen samples. The occurrence of the viruses was examined against the variables of urban index, air pollution (O3 and NO2), air temperature, and tree morphometrics (trunk perimeter, tree height, crown height and diameter). Generalized Non-linear models (binomial logit with backward stepwise removal of independent variables) were employed. During the study period, both CLRV and BIV were distributed widely throughout the investigated birch individuals. CLRV seemed to be rather cosmopolitan and was present independent of any abiotic factor. BIV's occurrence was mostly determined by higher values of the urban index and of NO2. Urban birch trees, located next to high-traffic roads with higher NO2 levels, are more likely to be infected by BIV. Increased environmental stress may lead to more plant viral infections. Here we suggest that this is particularly true for urban spaces, near high-traffic roads, where plants may be more stressed, and we recommend taking mitigation measures for controlling negative human interventions.

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.

Molecular characterization of horse nettle virus A, a new member of subgroup B of the genus Nepovirus.

A new positive-strand RNA virus was discovered in a horse nettle plant, using high-throughput sequencing (HTS), and its complete genome, consisting of RNA1 and RNA2, which are 7522 and 4710 nucleotides in length, respectively, was characterized. Each genome segment contains a single open reading frame flanked by 5' and 3' untranslated regions (UTRs), followed by a poly(A) tail at the 3' end. The encoded proteins have the highest amino acid sequence identity (55% and 45%) to the polyprotein encoded by RNA1 of tomato black ring virus (TBRV) and RNA2 of potato virus B (PVB), respectively. Its genome organization and phylogenetic relationship to other nepoviruses suggested that this virus is a novel member of subgroup B, and recombination analysis revealed its evolutionary history within the subgroup. These results suggest the new virus, provisionally named "horse nettle virus A", represents a new species within the genus Nepovirus.

Characterization of Grapevine Fanleaf Virus Isolates in 'Chardonnay' Vines Exhibiting Severe and Mild Symptoms in Two Vineyards.

Fanleaf degeneration is a complex viral disease of Vitis spp. that detrimentally impacts fruit yield and reduces the productive lifespan of most vineyards worldwide. In France, its main causal agent is grapevine fanleaf virus (GFLV). In the past, field experiments were conducted to explore cross-protection as a management strategy of fanleaf degeneration, but results were unsatisfactory because the mild virus strain negatively impacted fruit yield. In order to select new mild GFLV isolates, we examined two old 'Chardonnay' parcels harbouring vines with distinct phenotypes. Symptoms and agronomic performances were monitored over the four-year study on 21 individual vines that were classified into three categories: asymptomatic GFLV-free vines, GFLV-infected vines severely diseased and GFLV-infected vines displaying mild symptoms. The complete coding genomic sequences of GFLV isolates in infected vines was determined by high-throughput sequencing. Most grapevines were infected with multiple genetically divergent variants. While no specific molecular features were apparent for GFLV isolates from vines displaying mild symptoms, a genetic differentiation of GFLV populations depending on the vineyard parcel was observed. The mild symptomatic grapevines identified during this study were established in a greenhouse to recover GFLV variants of potential interest for cross-protection studies.

Re-examination of nepovirus polyprotein cleavage sites highlights the diverse specificities and evolutionary relationships of nepovirus 3C-like proteases.

Plant-infecting viruses of the genus Nepovirus (subfamily Comovirinae, family Secoviridae, order Picornavirales) are bipartite positive-strand RNA viruses with each genomic RNA encoding a single large polyprotein. The RNA1-encoded 3C-like protease cleaves the RNA1 polyprotein at five sites and the RNA2 polyprotein at two or three sites, depending on the nepovirus. The specificity of nepovirus 3C-like proteases is notoriously diverse, making the prediction of cleavage sites difficult. In this study, the position of nepovirus cleavage sites was systematically re-evaluated using alignments of the RNA1 and RNA2 polyproteins, phylogenetic relationships of the proteases, and sequence logos to examine specific preferences for the P6 to P1' positions of the cleavage sites. Based on these analyses, the positions of previously elusive cleavage sites, notably the 2a-MP cleavage sites of subgroup B nepoviruses, are now proposed. Distinct nepovirus protease clades were identified, each with different cleavage site specificities, mostly determined by the nature of the amino acid at the P1 and P1' positions of the cleavage sites, as well as the P2 and P4 positions. The results will assist the prediction of cleavage sites for new nepoviruses and help refine the taxonomy of nepoviruses. An improved understanding of the specificity of nepovirus 3C-like proteases can also be used to investigate the cleavage of plant proteins by nepovirus proteases and to understand their adaptation to a broad range of hosts.

Genetic Diversity of Tomato Black Ring Virus Satellite RNAs and Their Impact on Virus Replication.

Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different isolates of tomato black ring virus (TBRV). We checked the presence of satRNAs in 42 samples infected with TBRV, performed recombination and genetic diversity analyses, and examined the selective pressure affecting the satRNAs population. We identified 18 satRNAs in total that differed in length and the presence of point mutations. Moreover, we observed a strong effect of selection operating upon the satRNA population. We also constructed infectious cDNA clones of satRNA and examined the viral load of different TBRV isolates in the presence and absence of satRNAs, as well as the accumulation of satRNA molecules on infected plants. Our data provide evidence that the presence of satRNAs significantly affects viral load; however, the magnitude of this effect differs among viral isolates and plant hosts. We also showed a positive correlation between the number of viral genomic RNAs (gRNAs) and satRNAs for two analysed TBRV isolates.

A novel nepovirus causing a chlorotic fleck disease on common bean (Phaseolus vulgaris L.).

Two common bean leaf samples from Ethiopia that had shown chlorotic fleck and veinal mosaic symptoms but tested ELISA-negative for known viruses were mechanically transmitted to herbaceous hosts to obtain virus isolates ET-773/4 and ET-779. Virus purification from Chenopodium quinoa systemically infected with ET-773/4 yielded icosahedral particles measuring ~ 30 nm in diameter and containing a single capsid protein of ~ 58 kDa, suggesting a nepovirus infection. Analysis of nucleotide sequences generated from RNA1 and RNA2 of the isolates indicated that they represent a distinct virus species in the genus Nepovirus. Surprisingly, the most closely related sequence in the GenBank database was that of Hobart nepovirus 3, an incompletely described metagenomic sequence obtained from honey bees in Tasmania. This new nepovirus from Ethiopia is provisionally named "bean chlorotic fleck virus".

Detection of Ampelovirus and Nepovirus by Lab-on-a-Chip: A Promising Alternative to ELISA Test for Large Scale Health Screening of Grapevine.

The Ampelovirus Grapevine leafroll-associated virus 3 (GLRaV-3) and the Nepovirus Grapevine fanleaf virus (GFLV) are pathogens reported in many grapevine-growing areas all over the world, main causal agents of grapevine leafroll disease and grapevine fanleaf disease, respectively. Prevention of virus spread thanks to rapid diagnosis of infected plants is a key factor for control of both diseases. Although serological (e.g., enzyme-linked immunosorbent assay-ELISA test) and molecular methods are available to reveal the presence of the viruses, they turn out to be quite expensive, time-consuming and laborious, especially for large-scale health screening. Here we report the optimization of a lab-on-a-chip (LOC) for GLRaV-3 and GFLV detection, based on an electrochemical transduction and a microfluidic multichamber design for measurements in quadruplicate and simultaneous detection of both targets. The LOC detect GLRaV-3 and GFLV at dilution factors more than 15 times higher than ELISA, providing a higher sensitivity in the detection of both viruses. Furthermore, the platform offers several advantages as easy-to-use, rapid-test, portability and low costs, favoring its potential application for large-scale monitoring programs. Compared to other grapevine virus biosensors, our sensing platform is the first one to provide a dose-dependent calibration curve combined with a microfluidic module for sample analysis and a portable electronics providing an operator-independent read-out scheme.

Population genetics of cycas necrotic stunt virus and the development of multiplex RT-PCR diagnostics.

Cycas necrotic stunt virus (CNSV) has an extensive host range and is detected in an accelerated pace around the globe in several agricultural crops. One of the plant species affected is peony (Paeonia lactiflora Pall.). The virus is asymptomatic in most peony cultivars, but there have been reports of symptoms in others. It is thus important to study CNSV and its population structure to gain insights into its evolution and epidemiology. The outputs of this study, in addition to the in-depth analysis of the virus population structure, include the development of a multiplex RT-PCR detection protocol that can amplify all published CNSV isolate sequences; allowing for accurate, reliable detection of the virus and safeguarding its susceptible, clonally-propagated hosts.

Severe Stunting Symptoms upon Nepovirus Infection Are Reminiscent of a Chronic Hypersensitive-like Response in a Perennial Woody Fruit Crop.

Virus infection of plants can result in various degrees of detrimental impacts and disparate symptom types and severities. Although great strides have been made in our understanding of the virus-host interactions in herbaceous model plants, the mechanisms underlying symptom development are poorly understood in perennial fruit crops. Grapevine fanleaf virus (GFLV) causes variable symptoms in most vineyards worldwide. To better understand GFLV-grapevine interactions in relation to symptom development, field and greenhouse trials were conducted with a grapevine genotype that exhibits distinct symptoms in response to a severe and a mild strain of GFLV. After validation of the infection status of the experimental vines by high-throughput sequencing, the transcriptomic and metabolomic profiles in plants infected with the two viral strains were tested and compared by RNA-Seq and LC-MS, respectively, in the differentiating grapevine genotype. In vines infected with the severe GFLV strain, 1023 genes, among which some are implicated in the regulation of the hypersensitive-type response, were specifically deregulated, and a higher accumulation of resveratrol and phytohormones was observed. Interestingly, some experimental vines restricted the virus to the rootstock and remained symptomless. Our results suggest that GFLV induces a strain- and cultivar-specific defense reaction similar to a hypersensitive reaction. This type of defense leads to a severe stunting phenotype in some grapevines, whereas others are resistant. This work is the first evidence of a hypersensitive-like reaction in grapevine during virus infection.

Genome sequence and geographic distribution of a new nepovirus infecting Stenotaphrum secundatum in Australia.

The genome sequence of a new subgroup C nepovirus from Stenotaphrum secundatum in Australia is described. This virus, tentatively named Stenotaphrum nepovirus (SteNV), was present in separate plants as a mixed infection with either sugarcane mosaic virus or Panicum mosaic virus. The virus genome was divided between two RNA segments, 7,824 and 7,104 nucleotides (nt) in length, which each encode a single long polyprotein with putative 3C-like cysteine protease sites of the type H/G, H/S or L/S. The 3' untranslated region of RNA2, at 2,155 nt, is the longest observed for any subgroup C nepovirus. Phylogenetic analyses using protease-polymerase and coat protein amino acid alignments suggest that SteNV is most closely related to cherry leaf roll virus. Using a newly developed RT-PCR assay, this virus was detected at multiple localities in New South Wales, Queensland and Western Australia, and in a second host species, Digitaria didactyla. No consistent association between virus infection and symptoms could be established. The economic importance, pathogenicity and transmission of this novel virus species warrant further investigation.

Metagenomic analysis of nepoviruses: diversity, evolution and identification of a genome region in members of subgroup A that appears to be important for host range.

Data mining and metagenomic analysis of 277 open reading frame sequences of bipartite RNA viruses of the genus Nepovirus, family Secoviridae, were performed, documenting how challenging it can be to unequivocally assign a virus to a particular species, especially those in subgroups A and C, based on some of the currently adopted taxonomic demarcation criteria. This work suggests a possible need for their amendment to accommodate pangenome information. In addition, we revealed a host-dependent structure of arabis mosaic virus (ArMV) populations at a cladistic level and confirmed a phylogeographic structure of grapevine fanleaf virus (GFLV) populations. We also identified new putative recombination events in members of subgroups A, B and C. The evolutionary specificity of some capsid regions of ArMV and GFLV that were described previously and biologically validated as determinants of nematode transmission was circumscribed in silico. Furthermore, a C-terminal segment of the RNA-dependent RNA polymerase of members of subgroup A was predicted to be a putative host range determinant based on statistically supported higher π (substitutions per site) values for GFLV and ArMV isolates infecting Vitis spp. compared with non-Vitis-infecting ArMV isolates. This study illustrates how sequence information obtained via high-throughput sequencing can increase our understanding of mechanisms that modulate virus diversity and evolution and create new opportunities for advancing studies on the biology of economically important plant viruses.

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.