Grapevine fanleaf virus (GFLV) is the main causal agent of fanleaf degeneration, the most damaging viral disease of grapevine. GFLV is included in most grapevine certification programs that rely on ...robust diagnostic tools such as biological indexing, serological methods, and molecular techniques, for the identification of clean stocks. The emergence of high throughput sequencing (HTS) offers new opportunities for detecting GFLV and other viruses in grapevine accessions of interest. Here, two HTS-based methods,
, RNAseq and smallRNAseq (focusing on the 21 to 27 nt) were explored for their potential to characterize the virome of grapevine samples from two 30-year-old GFLV-infected vineyards in the Champagne region of France. smallrnaseq was optimal for the detection of a wide range of viral species within a sample and RNAseq was the method of choice for full-length viral genome assembly. The implementation of a protocol to discriminate between low GFLV titer and
contamination (intra-lane contamination due to index misassignment) during data processing was critical for data analyses. Furthermore, we compared the performance of semi-quantitative DAS-ELISA (double antibody enzyme-linked immunosorbent assay), RT-qPCR (Reverse transcription-quantitative polymerase chain reaction), Immuno capture (IC)-RT-PCR, northern blot for viral small interfering RNA (vsiRNA) detection and RNAseq for the detection and quantification of GFLV. While detection limits were variable among methods, as expected, GFLV diagnosis was consistently achieved with all of these diagnostic methods. Together, this work highlights the robustness of DAS-ELISA, the current method routinely used in the French grapevine certification program, for the detection of GFLV and offers perspectives on the potential of HTS as an approach of high interest for certification.
Since its identification in 2003, grapevine Pinot gris virus (GPGV,
Trichovirus
) has now been detected in most grape-growing countries. So far, little is known about the epidemiology of this newly ...emerging virus. In this work, we used datamining as a tool to monitor
in-silico
the sanitary status of three vineyards in Italy. All data used in the study were recovered from a work that was already published and for which data were publicly available as SRA (Sequence Read Archive, NCBI) files. While incomplete, knowledge gathered from this work was still important, with evidence of differential accumulation of the virus in grapevine according to year, location, and variety-rootstock association. Additional data regarding GPGV genetic diversity were collected. Some advantages and pitfalls of datamining are discussed.
Grapevine fanleaf virus (GFLV) is responsible for a widespread disease in vineyards worldwide. Its genome is composed of two single-stranded positive-sense RNAs, which both show a high genetic ...diversity. The virus is transmitted from grapevine to grapevine by the ectoparasitic nematode
. Grapevines in diseased vineyards are often infected by multiple genetic variants of GFLV but no information is available on the molecular composition of virus variants retained in
following nematodes feeding on roots. In this work, aviruliferous
were fed on three naturally GFLV-infected grapevines for which the virome was characterized by RNAseq. Six RNA-1 and four RNA-2 molecules were assembled segregating into four and three distinct phylogenetic clades of RNA-1 and RNA-2, respectively. After 19 months of rearing, single and pools of 30
tested positive for GFLV. Additionally, either pooled or single
carried multiple variants of the two GFLV genomic RNAs. However, the full viral genetic diversity found in the leaves of infected grapevines was not detected in viruliferous nematodes, indicating a genetic bottleneck. Our results provide new insights into the complexity of GFLV populations and the putative role of
as reservoirs of virus diversity.
Since its identification in 2003, little has been revealed about the spread of grapevine Pinot gris virus (GPGV), an emerging grapevine virus. According to studies from Italy, GPGV transmission in ...the vineyard can be fast but progressive over the years. To gain new insights into the spread of GPGV infections, we tested 67 grapevines in a single vineyard parcel in southern France. These vines were sampled over 8 years (2013 to 2020) and tested for GPGV by reverse-transcription PCR using a new primer pair designed from the recently described genetic diversity of GPGV worldwide. While focusing on a portion of the samples (n = 20), we observed a drastic increase in newly GPGV-infected vines from 2014 (5%, 1 of 20) to 2015 (80%, 16 of 20) and 2016 (90%, 18 of 20). Infected vines were scattered throughout the vineyard with no distinct pattern of distribution, and some rare vines remained negative through 2020. Using all available genomic information, we performed Bayesian-based phylogeographic analyses that identified a major intravineyard transmission in 2014 to 2015. To test our model, we analyzed 47 additional grapevines and confirmed the outbreak of GPGV in 2015, validating our in silico projection. Interestingly, some grapevines remained negative throughout the study, in spite of their close proximity to infected plants. These results raise questions about the dynamics of vector populations and environmental conditions that may be required for virus spread to occur in the vineyard.
Grapevine fanleaf virus (GFLV) and arabis mosaic virus (ArMV) are nepoviruses responsible for grapevine degeneration. They are specifically transmitted from grapevine to grapevine by two distinct ...ectoparasitic dagger nematodes of the genus
. GFLV and ArMV move from cell to cell as virions through tubules formed into plasmodesmata by the self-assembly of the viral movement protein. Five surface-exposed regions in the coat protein called R1 to R5, which differ between the two viruses, were previously defined and exchanged to test their involvement in virus transmission, leading to the identification of region R2 as a transmission determinant. Region R4 (amino acids 258 to 264) could not be tested in transmission due to its requirement for plant systemic infection. Here, we present a fine-tuning mutagenesis of the GFLV coat protein in and around region R4 that restored the virus movement and allowed its evaluation in transmission. We show that residues T258, M260, D261, and R301 play a crucial role in virus transmission, thus representing a new viral determinant of nematode transmission.
Factors involved in symptom expression of viruses from the genus Nepovirus in the family Secoviridae such as grapevine fanleaf virus (GFLV) are poorly characterized. To identify symptom determinants ...encoded by GFLV, infectious cDNA clones of RNA1 and RNA2 of strain GHu were developed and used alongside existing infectious cDNA clones of strain F13 in a reverse genetics approach. In vitro transcripts of homologous combinations of RNA1 and RNA2 induced systemic infection in Nicotiana benthamiana and Nicotiana clevelandii with identical phenotypes to WT virus strains, i.e. vein clearing and chlorotic spots on N. benthamiana and N. clevelandii for GHu, respectively, and lack of symptoms on both hosts for F13. The use of assorted transcripts mapped symptom determinants on RNA1 of GFLV strain GHu, in particular within the distal 408 nt of the RNA-dependent RNA polymerase (1E(Pol)), as shown by RNA1 transcripts for which coding regions or fragments derived thereof were swapped. Semi-quantitative analyses indicated no significant differences in virus titre between symptomatic and asymptomatic plants infected with various recombinants. Also, unlike the nepovirus tomato ringspot virus, no apparent proteolytic cleavage of GFLV protein 1E(Pol) was detected upon virus infection or transient expression in N. benthamiana. In addition, GFLV protein 1E(Pol) failed to suppress silencing of EGFP in transgenic N. benthamiana expressing EGFP or to enhance GFP expression in patch assays in WT N. benthamiana. Together, our results suggest the existence of strain-specific functional domains, including a symptom determinant module, on the RNA-dependent RNA polymerase of GFLV.
Grapevine virus A (GVA, Vitivirus) was transmitted experimentally by first and second instars of the scale insect Parthenolecanium corni from grapevine to grapevine and to the herbaceous host ...Nicotiana benthamiana. This is the first report of GVA transmission by P. corni. Grapevine leafroll-associated virus-1 (Ampelovirus) was always present in the donor grapevines and, in every case, GVA was transmitted simultaneously with this ampelovirus from grapevine to grapevine, suggesting possible interactions between the two viruses for transmission.
The viral determinants involved in the specific transmission of
Grapevine fanleaf virus (GFLV) by its nematode vector
Xiphinema index are located within the 513 C-terminal residues of the ...RNA2-encoded polyprotein, that is, the 9 C-terminal amino acids of the movement protein (2B
MP) and contiguous 504 amino acids of the coat protein (2C
CP) Virology 291 (2001) 161. To further delineate the viral determinants responsible for the specific spread, the four amino acids that are different within the 9 C-terminal 2B
MP residues between GFLV and
Arabis mosaic virus (ArMV), another nepovirus which is transmitted by
Xiphinema diversicaudatum but not by
X. index, were subjected to mutational analysis. Of the recombinant viruses derived from transcripts of GFLV RNA1 and RNA2 mutants that systemically infected herbaceous host plants, all with the 2C
CP of GFLV were transmitted by
X. index unlike none with the 2C
CP of ArMV, regardless of the mutations within the 2B
MP C-terminus. These results demonstrate that the coat protein is the sole viral determinant for the specific spread of GFLV by
X. index.
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