Grapevine Fanleaf Virus Isolates Research Articles

Real-time RT-PCR high-resolution melting curve analysis to detect and differentiate Brazilian variants of grapevine viruses

Detecting and identifying viral infections in perennial plants, such as grapevines, can be challenging. Therefore, the aim of this study was to perform a real-time RT-PCR (RT-qPCR) high-resolution melting (HRM) curve analysis to detect and differentiate Brazilian variants of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine fanleaf virus (GFLV) in 74 and 10 infected plants, respectively, maintained in a collection block of grapevines. A single amplification curve was generated for each sample by RT-qPCR. Considering the amplified region of genomes of these two viruses, it was possible to identify and distinguish different variants of GLRaV-3 and of GFLV, which showed significantly different melting temperature (Tm) values between themselves, reflecting differences in the nucleotide sequences of the respective amplicons, and allowing discriminating variants and assess the viral diversity in grapevine accessions. The HRM analysis was validated by sequencing and nucleotide comparisons among Brazilian isolates of GLRaV-3 and GFLV.

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.

1-Phylogenetic status of Grapevine fanleaf virus isolate of Lorestan province of Iran

1-Phylogenetic status of Grapevine fanleaf virus isolate of Lorestan province of Iran

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.

Association of satellite RNA with Grapevine fanleaf virus in its geographical origin and sequence characteristics

The region between The Caspian Sea and Black Sea has been hypothesized as the origin of grapevine. Likewise, an extensive study on Grapevine fanleaf virus (GFLV) from this region suggests this region as a possible origin of the virus. However, as yet there is no information as to whether or not the virus variants from this region accompanied with the virus satellite RNA (satRNA) and if so, how diverse theses satRNAs can be. To answer these questions, Grapevine fanleaf virus isolates were collected from different vineyards in the northwest region of Iran for the occurrence of the satellite RNA. A total of 421 samples including Vitis vinifera , Chenopodium quinoa , Chenopodium amaranticolor , Cynodon dactylis , Medicago sativa were initially screened against GFLV by RT-PCR with the coat protein primers (Cp433, Cp912). When the GFLV-infected plants (36 samples) were screened by RT-PCR with the newly designed satRNA primers Gf750 and Gr750, three samples appeared to possess the satRNA. The satellite cDNA fragments were cloned and sequenced and when the resulting data were compared with sequences of previously-reported satRNA of GFLV and Arabis mosaic virus (ArMV), 70–98% and 69–71% similarities were found, respectively. Phylogenetic studies revealed distinctness of the satRNAs from Iran. This may suggest coevolution of the satRNA with the helper virus because GFLV isolates from this region also form a distinct branch. Among previously reported corresponding satRNA sequences to those from Slovenia and South Central Europe were the closest. This study also reports C. amaranticolor as the natural host for GFLV, which was previously reported only as an experimental host.

Structural basis of nanobody recognition of grapevine fanleaf virus and of virus resistance loss

Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV-Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.

Detection and identification of a new isolate of Grapevine fanleaf Virus naturally infecting Grapevine plants in Egypt using qReal Time-PCR

Grapevine fanleaf virus (GFLV) is a member of the genus Nepovirus in the family Comoviridae, a widely distributed virus responsible for grapevine (Vitis vinifera) degeneration. This virus causes serious economic losses by reducing grape crop yield. The Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction (qReal Time-PCR) assay was carried out on (GFLV) recovered from infected grapevines leaves at Alexandria, Egypt. A 606 bp fragment of the GFLV RNA-2 coat protein (CP) gene was amplified and then sequenced. Results of reactions of diagnostic hosts were observed on Gomphrena globosa, which developed systemic mottling, leaves twisting and necrotic spots during spring, whereas Chenopodium amaranticolor induced systemic mottling and leaf deformation, and its sap seemed relatively insensitive to the inhibitors of infection. Mottling of Glycine max was detected after inoculation, but inoculation of Nicotiana glutinosa didn't induce any symptoms. This study aimed to detect and identify a new isolate of GFLV-DA3 from Egypt using biological and molecular tools.

Nanobody-mediated resistance to Grapevine fanleaf virus in plants.

SummarySince their discovery, single‐domain antigen‐binding fragments of camelid‐derived heavy‐chain‐only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode‐transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell‐to‐cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs.

Dynamics of the population structure and genetic variability within Iranian isolates of grapevine fanleaf virus: evidence for polyphyletic origin.

To determine the genetic diversity and population structure of grapevine fanleaf virus (GFLV), the complete nucleotide sequence of the coat protein gene of 41 isolates from different regions in Iran was determined. Phylogenetic analyses of these isolates together with those available in the GenBank revealed two evolutionary divergent lineages, designated GFLV-G and GFLV-Ir that reflect origin of the isolates. Analysis of the genetic variability in the coat protein of these isolates revealed 37 genotype groups in GFLV population. Analyses indicate that GFLV-G and GFLV-Ir clades are significantly differentiated populations of GFLV. Also, geographical subpopulations of the virus in Iran were completely distinct from each other. Examination of nonsynonymous/synonymous nucleotide diversity showed that the CP gene has been under purifying selection. The neutrality tests indicate balancing selection operating within isolates of the northwest of Iran and purifying selection within the other populations.

Complete nucleotide sequence of a new variant of grapevine fanleaf virus from northeastern China.

The complete RNA1 and RNA2 sequences of a new grapevine fanleaf virus isolate (GFLV-SDHN) from northeastern China were determined. The two RNAs are 7,367 and 3,788 nucleotides (nt) in length, respectively, excluding the poly(A) tails. Compared to other GFLV isolates, GFLV-SDHN has a 22- to 24-nt insertion in the RNA1 5' untranslated region, and there was 19.1-20.1% and 11.7%-13.0% sequence divergence in RNA1, and 15.5%-20.5% and 8.5-13.5% in RNA2, at the nt and amino acid level, respectively. Phylogenetic analysis revealed that the origins of GFLV-SDHN are distinct from those of other GFLV isolates. One recombination event was identified in the 2AHP region of RNA2 in GFLV-SDHN.

Survey of viral infections in spontaneous grapevines from natural environments in Sicily

The presence of nine of the most economical important grapevine viruses was surveyed in eight spontaneous grapevine populations from natural areas of Sicily. Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus 1, 2 and 3 (GLRaV-1, −2 and −3), Grapevine virus A and B (GVA, GVB), Grapevine rupestris stem pitting-associated virus (GRSPaV) and Grapevine fleck virus (GFKV) infections were assessed by molecular and serological methods in 73 vines collected in summer 2013 and winter 2013–2014. Reverse transcription-multiplex PCR detected nine GRSPaV- and one GFLV-infected plants in five and one grapevine populations, respectively, while no ArMV, GLRaV-1, GLRaV-2, GLRaV-3, GVA, GVB, GFKV infections were detected. ELISA tests on dormant wood confirmed the result of the molecular detection. The relationship of Sicilian wild grapevine isolates with isolates from grapevines of different geographic origin was investigated through sequencing of the complete coat protein (CP) region of GRSPaV, and partial CP and homing protein (HP) domains of GFLV. Pairwise comparison among the Sicilian GRSPaV CP sequences showed identity scores ranging from 81.67 % to 99.87 % and from 92.66 % to 100 % at nucleotide (nt) and amino acid level (aa), respectively. For GFLV, the CP sequence showed identity ranges from 84.28 % to 90.48 % (nt) and from 89.39 % to 97.11 % (aa); higher variability was obtained analysing the HP domain with identity scores ranging from 68.42 to 93.28 % (nt) and from 58.02 % to 93.23 % (aa). According to phylogenetic analyses, GRSPaV and GFLV isolates from Sicilian wild grapevines clustered with isolates from cultivated grapevines without any correlation between isolate distribution and their geographic origin.

Detection and genetic variation analysis of grapevine fanleaf virus (GFLV) isolates in China.

To investigate the prevalence and genetic variation of grapevine fanleaf virus (GFLV) in China, 142 grapevine samples from 13 provinces and regions were tested using DAS-ELISA, RT-PCR, and nested RT-PCR. Of the samples, 38% tested positive for GFLV by DAS-ELISA, and 26.8% tested positive by RT-PCR and nested RT-PCR. Movement protein (MP) and coat protein (CP) gene PCR products were cloned and sequenced. The MP or CP nucleotide and protein sequences shared identities that ranged from 94.9% to 100%. Phylogenetic analysis revealed that Chinese GFLV isolates obtained in this study were distinct from the isolates reported in GenBank.

Genetic variability of the movement and coat protein genes of Grapevine fanleaf virus isolates from Italy

Grapevine fanleaf virus (GFLV), a nematode-transmitted virus belonging to the genus Nepovirus, is the major pathogen responsible for fanleaf degeneration, one of the most widespread and damaging viral diseases of grapevine. The virus is characterized by a genome constituted by two single positive sense RNAs (RNA1 and RNA2), coding for two polyproteins. Here we investigated the genetic variability of the movement and coat protein genes (2BMP and 2CCP) of Italian GFLV isolates by sequencing analyses. The presence of high molecular heterogeneity between our isolates suggests that GFLV comprises a family of sequence variants.

The demonstration of the GFLV Nepovirus isolates on naturally infected grapevine cultivars and evaluation of variability within genome region encoding movement protein

In this work, the infection of Grapevine fanleaf virus (GFLV) disease was described on the base of symptomatic differences within eight grapevine plants of six grape ciltivars with positive tests on GFLV. Among them, cultivars Kodrjanka, Pamjati Negrula, Kišmiš Lučistyj were planted in wine region of the South Moravia (Czech Republic), three infected grapevine cultivars (URS, Cinsaut, Dimrit) included in this study originating from Italy. Except symptomatic evaluation, the differences between isolates were emphasized at the genetic level too, exactly in the frame of RNA2 genomic region coding movement protein. The variability of the tested isolates within the eight plants was in the range from 86.59 to 97.61% at the nucleotide level. The results confirmed very high degree of similarity between virus isolates of GFLV within studied RNA2 region. This fact was assessed by the phylogenetic analysis of obtained sequencing data too.

Phylogenetic and recombination analysis of the homing protein domain of grapevine fanleaf virus (GFLV) isolates associated with 'yellow mosaic' and 'infectious malformation' syndromes in grapevine.

The RNA2 of seven grapevine fanleaf virus (GFLV) isolates from vines with yellow mosaic (YM) symptoms from different origin were sequenced. These sequences showed a high variability in the homing protein (2A(HP)) and, in five of them, a putative recombination with arabis mosaic virus (ArMV) was detected. To investigate recombination frequency, the partial sequences of the 2A(HP) of 28 additional GFLV isolates from nine different countries, showing either YM or infectious malformations (MF) symptoms, were obtained and compared with those of GFLV isolates from GenBank. The analysis confirmed the high level of sequence variability (up to 41 % at the nucleotide level) among isolates. In phylogenetic trees constructed using different approaches, the sequenced isolates always clustered in four conserved groups, three of which comprised YM strains (groups 1, 2 and 3), and one (group 4) the MF strains. Potential interspecific recombination sites between GFLV and ArMV were predicted in the 2A(HP) gene of several isolates, all of which were associated with YM symptoms.

Detection of Grapevine fanleaf virus by immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) with recombinant antibody

Grapevine fanleaf virus (GFLV), a member of the genus Nepovirus in the family Secoviridae, is the most widely distributed grapevine virus around the world. In the present research, recombinant antibody to the GFLV coat protein (CP) was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) for the detection of GFLV by the use of a pair of primers corresponding to the full length CP gene. In addition, double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was done by the use of recombinant antibody to detect the GFLV isolates. Eventually, an expected band with around 1512 base pair (bp) in length was revealed for three isolates by IC-RT-PCR. Also, DAS-ELISA revealed that the recombinant antibody was able to detect the virus in leaf sample. These results showed that application of recombinant antibody in these detection methods can be useful for rapid detection of viruses in plant tissue because by applying IC-RT-PCR along with recombinant antibody, it is possible to overcome the limitations happened by presence of inhibitors in grapevine trees and non-specific reaction resulting from presence of native proteins in plant sap against conventionally made antibody.

Complete Nucleotide Sequence of a South African Isolate of Grapevine Fanleaf Virus and Its Associated Satellite RNA

The complete sequences of RNA1, RNA2 and satellite RNA have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SACH44). The two RNAs of GFLV-SACH44 are 7,341 nucleotides (nt) and 3,816 nt in length, respectively, and its satellite RNA (satRNA) is 1,104 nt in length, all excluding the poly(A) tail. Multiple sequence alignment of these sequences showed that GFLV-SACH44 RNA1 and RNA2 were the closest to the South African isolate, GFLV-SAPCS3 (98.2% and 98.6% nt identity, respectively), followed by the French isolate, GFLV-F13 (87.3% and 90.1% nt identity, respectively). Interestingly, the GFLV-SACH44 satRNA is more similar to three Arabis mosaic virus satRNAs (85%–87.4% nt identity) than to the satRNA of GFLV-F13 (81.8% nt identity) and was most distantly related to the satRNA of GFLV-R2 (71.0% nt identity). Full-length infectious clones of GFLV-SACH44 satRNA were constructed. The infectivity of the clones was tested with three nepovirus isolates, GFLV-NW, Arabis mosaic virus (ArMV)-NW and GFLV-SAPCS3. The clones were mechanically inoculated in Chenopodium quinoa and were infectious when co-inoculated with the two GFLV helper viruses, but not when co-inoculated with ArMV-NW.

Short communication. Molecular analysis of the genomic RNAs 1 and 2 of the first Arabis mosaic virus isolate detected in Spanish grapevines

The Arabis mosaic virus (ArMV) is one of the causative agent of the grapevine fanleaf disease, one of the most widespread and damaging viral diseases of grapevine. Recently, the ArMV has been detected in Spanish vineyards, and its determination and molecular characterization was undertaken. To this aim, the nucleotide sequence of the genomic RNAs 1 and 2 of the first isolate of ArMV infecting grapevine detected in Spain (ArMV-DU13) has been determined. The ArMV-DU13 genomic sequences were compared to the corresponding sequences of other isolates of ArMV, or nepoviruses. The most divergent genes among ArMV isolates were the X1 and VPg genes on the RNA 1, and the 2A gene on the RNA 2, with identity levels at the amino acid level of 78% (X1 and VPg) or 69% (2A) between the most distant isolates. Interestingly, the VPg genes were identical between the two grapevine isolates ArMV-Du13 and –NW, suggesting a possible implication of the host. The phylogenetic analysis of the RNA 2 showed that the Spanish isolate was close to Grapevine fanleaf virus isolates. The analysis of the full length RNA 2 suggests a recombination event between ArMV-DU13 and GFLV-GHu isolates between nucleotides 54 and 586 in the ArMV-DU13 isolate. Altogether, these results confirm the high variability between isolates of ArMV, and will be helpful to design more appropriate and reliable molecular diagnostic techniques for the control of this emerging virus in Spain.

Population genetic analysis of grapevine fanleaf virus

Population genetic analysis of grapevine fanleaf virus (GFLV) was done on the basis of the virus movement protein (MP) gene sequences from the isolates detected and identified in this study and those of all previously reported GFLV strains/isolates. These revealed that the GFLV populations of Iran and Slovenia were highly distinct, whereas those of France, Germany, Italy and the USA were composed of multiple lineages. All populations were significantly differentiated from each other. However, two GFLV isolates from Tunisia, the only recorded GFLVs from that country, were not statistically distinct from the French, German and Italian populations. The ratio of non-synonymous nucleotide diversity to synonymous nucleotide diversity (Pi(a)/Pi(s)) was less than 1, suggesting that the MP gene has been under purifying selection. The neutrality tests were indicative of a balancing selection that is operating within Iranian and USA GFLV isolates, but they show a purifying selection within the other populations. Eleven recombination events were detected in a total of 50 isolates from France, Germany, Iran, Italy, Slovenia and the USA. The results from the recombination analysis were in agreement with those of the phylogenetic analysis. This study suggests that diversity among GFLV geographical populations resulted from possible host adaptation, recombination and founder effects.

Complete nucleotide sequence of a South African isolate of Grapevine fanleaf virus

The complete sequences of RNA1 and RNA2 have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SAPCS3). The two RNAs are, respectively, 7,342 and 3,817 nucleotides in length, excluding the poly(A) tails. RNA1 has a large open reading frame (ORF) of 6,852 nucleotides and a 5'-UTR and a 3'-UTR of 243 and 244 nucleotides, respectively. RNA2 encodes for an ORF of 3,330 nucleotides and has the highest nucleotide identity (90.4 %) with GFLV-F13. The full length nucleotide sequence of GFLV-SAPCS3 RNA1 had the highest nucleotide identity (86.5 %) to the French isolate GFLV-F13. The 5'- and 3'-UTRs of GFLV-SAPCS3 RNA2 are 272 nucleotides and 212 nucleotides (nt) in length, respectively. The GFLV-SAPCS3 RNA2 5'-UTR is 32-53 nt longer compared to other GFLV isolates. The GFLV-SAPCS3 RNA2 5'-UTR is also more closely related to GFLV-GHu and Arabis mosaic virus (ArMV) isolates than to other GFLV isolates. Putative intra- and interspecies recombination events between GFLV and ArMV isolates involving GFLV-SAPCS3 RNA1 and RNA2 were investigated. Recombination analysis software has indicated that the GFLV-SAPCS3 5'-UTR might have evolved from a recombinational event between GFLV-F13-type and ArMV-Ta-type isolate.