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1.
Arch Virol ; 166(10): 2789-2801, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34370094

RESUMO

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.


Assuntos
Genoma Viral/genética , Especificidade de Hospedeiro/genética , Nepovirus/genética , Evolução Molecular , Variação Genética , Metagenômica , Nepovirus/classificação , Fases de Leitura Aberta/genética , Filogenia , Filogeografia , Plantas/classificação , Plantas/virologia , RNA Viral/genética , Recombinação Genética
2.
Commun Biol ; 4(1): 637, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050254

RESUMO

Grapevine fanleaf disease, caused by grapevine fanleaf virus (GFLV), transmitted by the soil-borne nematode Xiphinema index, provokes severe symptoms and economic losses, threatening vineyards worldwide. As no effective solution exists so far to control grapevine fanleaf disease in an environmentally friendly way, we investigated the presence of resistance to GFLV in grapevine genetic resources. We discovered that the Riesling variety displays resistance to GFLV, although it is susceptible to X. index. This resistance is determined by a single recessive factor located on grapevine chromosome 1, which we have named rgflv1. The discovery of rgflv1 paves the way for the first effective and environmentally friendly solution to control grapevine fanleaf disease through the development of new GFLV-resistant grapevine rootstocks, which was hitherto an unthinkable prospect. Moreover, rgflv1 is putatively distinct from the virus susceptibility factors already described in plants.


Assuntos
Resistência à Doença/genética , Nepovirus/patogenicidade , Vitis/genética , Agricultura/métodos , Animais , Genótipo , Nematoides/virologia , Nepovirus/genética , Melhoramento Vegetal/métodos , Doenças das Plantas/genética , Doenças das Plantas/virologia , Vitis/metabolismo , Vitis/microbiologia
3.
J Gen Virol ; 102(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34043500

RESUMO

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.


Assuntos
Nepovirus/fisiologia , Mapas de Interação de Proteínas , RNA Polimerase Dependente de RNA/metabolismo , Tabaco/virologia , Proteínas Virais/metabolismo , Agrobacterium tumefaciens/genética , Cromatografia de Afinidade , Interações Hospedeiro-Patógeno , Mutação , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Proteômica , RNA Polimerase Dependente de RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas em Tandem , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
4.
PLoS One ; 16(4): e0249928, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33836032

RESUMO

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5'-viral genome-linked protein (VPg) and have a 3' polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3' untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5' regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5' region and 3' UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5' region and 3' UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.


Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Nepovirus/genética , Capuzes de RNA/fisiologia , RNA Viral/biossíntese , Sequência de Bases , Códon de Iniciação , Genes Reporter , Lycopersicon esculentum/virologia , Mutagênese , RNA Viral/genética , Alinhamento de Sequência
5.
Viruses ; 13(2)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33562555

RESUMO

Viral diseases in viticulture lead to annual losses in the quantity and quality of grape production. Since no direct control measures are available in practice, preventive measures are taken to keep the vines healthy. These include, for example, the testing of propagation material for viruses such as Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV) or Grapevine leafroll-associated virus 1 (GLRaV-1) and 3 (GLRaV-3). As long-term investigations have shown, GLRaV-1 (2.1%) occurs most frequently in southwestern German wine-growing regions, whereas GLRaV-3 (<0.1%) is almost never found. However, tests conducted over 12 years indicate that there is no general decline in virus-infected planting material. Thus, it can be assumed that a spread of the viruses via corresponding vectors still takes place unhindered. Beyond the examinations regulated within the German Wine Growing Ordinance, one-time tests were carried out on Grapevine Pinot gris virus (GPGV). This analysis showed that GPGV was found in 17.2% of the samples.


Assuntos
Closteroviridae/isolamento & purificação , Nepovirus/isolamento & purificação , Doenças das Plantas/virologia , Tymoviridae/isolamento & purificação , Vitis/virologia , Ensaio de Imunoadsorção Enzimática , Alemanha , Vinho
6.
Plant Dis ; 105(5): 1432-1439, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33048594

RESUMO

In 2012, dormant canes of a proprietary wine grape (Vitis vinifera L.) accession were included in the collection of the University of California-Davis Foundation Plant Services. No virus-like symptoms were elicited when bud chips from propagated own-rooted canes of the accession were graft-inoculated onto a panel of biological indicators. However, chlorotic ringspot symptoms were observed on sap-inoculated Chenopodium amaranticolor Coste & A. Rein and C. quinoa Willd. plants, indicating the presence of a mechanically transmissible virus. Transmission electron microscopy of virus preparations from symptomatic C. quinoa revealed spherical, nonenveloped virions about 27 nm in diameter. Nepovirus-like haplotypes of sequence contigs were detected in both the source grape accession and symptomatic C. quinoa plants via high-throughput sequencing. A novel bipartite nepovirus-like genome was assembled from these contigs, and the termini of each RNA segment were verified by rapid amplification of complementary DNA ends assays. The RNA1 (7,186-nt) of the virus encodes a large polyprotein 1 of 231.1 kDa, and the RNA2 (4,460-nt) encodes a large polyprotein 2 of 148.9 kDa. Each of the polyadenylated RNA segments is flanked by 5'- (RNA1 = 156-nt; RNA2 = 170-nt) and 3'- (RNA1 = 834-nt; RNA2 = 261-nt) untranslated region sequences with >90% identities. Maximum likelihood phylogenetic analyses of the conserved Pro-Pol amino acid sequences revealed the clustering of the new virus within the genus Nepovirus of the family Secoviridae. Considering its biological and molecular characteristics, and based on current taxonomic criteria, we propose that the novel virus, named grapevine nepovirus A, be assigned to the genus Nepovirus.


Assuntos
Nepovirus , Vitis , Nepovirus/genética , Filogenia , Poliproteínas , RNA Viral/genética
7.
Methods Mol Biol ; 2172: 123-138, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32557366

RESUMO

Virus-induced gene silencing (VIGS) is a powerful reverse genetic tool for rapid functional analysis of plant genes. Over the last decade, VIGS has been widely used for conducting rapid gene knockdown experiment in plants and played a crucial role in advancing applied and basic research in plant science. VIGS was studied extensively in model plants Arabidopsis and tobacco. Moreover, several non-model plants such as Papaver (Hileman et al., Plant J 44:334-341, 2005), Aquilegia (Gould and Kramer, Plant Methods 3:6, 2007), Catharanthus (Liscombe and O'Connor, Phytochemistry 72:1969-1977, 2011), Withania (Singh et al., Plant Biol J 13:1287-1299, 2015), and Ocimum (Misra et al., New Phytol 214:706-720, 2017) were also successfully explored. We have recently developed a robust protocol for VIGS in sweet basil (Ocimum basilicum). Sweet basil, a popular medicinal/aromatic herb, is being studied for the diversity of specialized metabolites produced in it.


Assuntos
Ocimum basilicum/metabolismo , Vírus de Plantas/patogenicidade , Agrobacterium/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genômica/métodos , Nepovirus/patogenicidade , Ocimum basilicum/genética
8.
Proc Natl Acad Sci U S A ; 117(20): 10848-10855, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32371486

RESUMO

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.


Assuntos
Nepovirus/efeitos dos fármacos , Doenças das Plantas/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacologia , Animais , Anticorpos Antivirais/imunologia , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/efeitos dos fármacos , Microscopia Crioeletrônica , Epitopos/química , Modelos Moleculares , Nematoides/virologia , Nepovirus/ultraestrutura , Doenças das Plantas/virologia , Folhas de Planta/virologia , Vírus de Plantas/imunologia , Vírus de Plantas/fisiologia , Conformação Proteica , Vitis
9.
BMC Plant Biol ; 20(1): 213, 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398088

RESUMO

BACKGROUND: Muscadine (Muscadinia rotundifolia) is known as a resistance source to many pests and diseases in grapevine. The genetics of its resistance to two major grapevine pests, the phylloxera D. vitifoliae and the dagger nematode X. index, vector of the Grapevine fanleaf virus (GFLV), was investigated in a backcross progeny between the F1 resistant hybrid material VRH8771 (Vitis-Muscadinia) derived from the muscadine R source 'NC184-4' and V. vinifera cv. 'Cabernet-Sauvignon' (CS). RESULTS: In this pseudo-testcross, parental maps were constructed using simple-sequence repeats markers and single nucleotide polymorphism markers from a GBS approach. For the VRH8771 map, 2271 SNP and 135 SSR markers were assembled, resulting in 19 linkage groups (LG) and an average distance between markers of 0.98 cM. Phylloxera resistance was assessed by monitoring root nodosity number in an in planta experiment and larval development in a root in vitro assay. Nematode resistance was studied using 10-12 month long tests for the selection of durable resistance and rating criteria based on nematode reproduction factor and gall index. A major QTL for phylloxera larval development, explaining more than 70% of the total variance and co-localizing with a QTL for nodosity number, was identified on LG 7 and designated RDV6. Additional QTLs were detected on LG 3 (RDV7) and LG 10 (RDV8), depending on the in planta or in vitro experiments, suggesting that various loci may influence or modulate nodosity formation and larval development. Using a Bulked Segregant Analysis approach and a proportion test, markers clustered in three regions on LG 9, LG 10 and LG 18 were shown to be associated to the nematode resistant phenotype. QTL analysis confirmed the results and QTLs were thus designated respectively XiR2, XiR3 and XiR4, although a LOD-score below the significant threshold value was obtained for the QTL on LG 18. CONCLUSIONS: Based on a high-resolution linkage map and a segregating grapevine backcross progeny, the first QTLs for resistance to D. vitifoliae and to X. index were identified from a muscadine source. All together these results open the way to the development of marker-assisted selection in grapevine rootstock breeding programs based on muscadine derived resistance to phylloxera and to X. index in order to delay GFLV transmission.


Assuntos
Resistência à Doença/genética , Hemípteros/fisiologia , Nematoides/fisiologia , Nepovirus/fisiologia , Doenças das Plantas/imunologia , Vitis/genética , Animais , Cruzamento , Mapeamento Cromossômico , Ligação Genética , Genótipo , Escore Lod , Repetições de Microssatélites/genética , Nematoides/virologia , Fenótipo , Doenças das Plantas/parasitologia , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Vitis/imunologia , Vitis/parasitologia
10.
J Virol Methods ; 278: 113821, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31958468

RESUMO

Raspberry ringspot virus (RpRSV) is an important virus that infects horticultural crops including grapevine, cherry, berry fruit and rose. The genome sequences of RpRSV are highly diverse between isolates and this makes the design of a PCR-based detection method difficult. In this study, a TaqMan real-time RT-PCR assay was developed for the rapid and sensitive detection of RpRSV. Primers and probes targeting the most conserved region of the movement protein gene were designed to amplify a 229 bp fragment of RpRSV RNA-2. The assay was able to amplify all RpRSV isolates tested. The detection limit of the RpRSV target region was estimated to be 61-98 copies, depending on the RpRSV strain. The sensitivity was about 100 times greater than the conventional RT-PCR assay using the same primers as the real-time RT-PCR assay. A comparison with published conventional RT-PCR assays indicated that both published assays lacked reliability and sensitivity, as neither were able to amplify all RpRSV isolates tested, and both were at least 1000 times less sensitive than the novel TaqMan real-time RT-PCR assay. The assay can also be run as a duplex reaction with the nad5 plant internal control primers and probe to simultaneously verify the PCR competency of the samples. The amplicon obtained with the real-time RT-PCR assay is suitable for direct sequencing if it is necessary to further confirm the RpRSV identity or determine the RpRSV strain.


Assuntos
Nepovirus/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rubus/virologia , Primers do DNA/genética , Limite de Detecção , Nepovirus/classificação , Doenças das Plantas/virologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
11.
Viruses ; 11(12)2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31835488

RESUMO

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 Xiphinema index. 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 X. index following nematodes feeding on roots. In this work, aviruliferous X. index 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 X. index tested positive for GFLV. Additionally, either pooled or single X. index 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 X. index as reservoirs of virus diversity.


Assuntos
Vetores de Doenças , Variação Genética , Nematoides/virologia , Nepovirus/genética , Vitis/parasitologia , Vitis/virologia , Animais , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Doenças das Plantas/virologia , RNA Viral
12.
Viruses ; 11(12)2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31835698

RESUMO

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 Xiphinema. 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.


Assuntos
Vetores de Doenças , Nematoides/virologia , Nepovirus/classificação , Nepovirus/fisiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/virologia , Sequência de Aminoácidos , Animais , Genes Reporter , Modelos Moleculares , Nepovirus/ultraestrutura , Conformação Proteica , RNA Viral , Recombinação Genética , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/genética
13.
BMC Biotechnol ; 19(1): 81, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31752839

RESUMO

BACKGROUND: Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. RESULTS: The epitope sequence was genetically inserted in the αB-αB" domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. CONCLUSIONS: The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.


Assuntos
Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/virologia , Humanos , Microscopia Eletrônica , Nepovirus/imunologia , Nepovirus/patogenicidade , Papillomaviridae/imunologia , Papillomaviridae/patogenicidade , Dobramento de Proteína
14.
Viruses ; 11(8)2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370205

RESUMO

An emerging virus-like flower yellowing disease (FYD) of green Sichuan pepper (Zanthoxylum armatum v. novemfolius) has been recently reported. Four new RNA viruses were discovered in the FYD-affected plant by the virome analysis using high-throughput sequencing of transcriptome and small RNAs. The complete genomes were determined, and based on the sequence and phylogenetic analysis, they are considered to be new members of the genera Nepovirus (Secoviridae), Idaeovirus (unassigned), Enamovirus (Luteoviridae), and Nucleorhabdovirus (Rhabdoviridae), respectively. Therefore, the tentative names corresponding to these viruses are green Sichuan pepper-nepovirus (GSPNeV), -idaeovirus (GSPIV), -enamovirus (GSPEV), and -nucleorhabdovirus (GSPNuV). The viral population analysis showed that GSPNeV and GSPIV were dominant in the virome. The small RNA profiles of these viruses are in accordance with the typical virus-plant interaction model for Arabidopsis thaliana. Rapid and sensitive RT-PCR assays were developed for viral detection, and used to access the geographical distributions. The results revealed a correlation between GSPNeV and the FYD. The viruses pose potential threats to the normal production of green Sichuan pepper in the affected areas due to their natural transmission and wide spread in fields. Collectively, our results provide useful information regarding taxonomy, transmission and pathogenicity of the viruses as well as management of the FYD.


Assuntos
Genoma Viral , Filogenia , Doenças das Plantas/virologia , Vírus de Plantas/classificação , Vírus de RNA/genética , Zanthoxylum/virologia , Luteoviridae/classificação , Luteoviridae/isolamento & purificação , Nepovirus/classificação , Nepovirus/isolamento & purificação , Fases de Leitura Aberta , Vírus de Plantas/isolamento & purificação , Vírus de RNA/isolamento & purificação , RNA Viral/genética , Rhabdoviridae/classificação , Rhabdoviridae/isolamento & purificação
15.
Virus Genes ; 55(5): 734-737, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31352619

RESUMO

Complete genome sequences of two cycas necrotic stunt virus (CNSV) isolates from Paeonia suffruticosa and Daphne odora were determined. Phylogenetic trees and pairwise comparisons using complete RNA1- and RNA2-encoded polyproteins showed that the two CNSV isolates are divergent (83.19%-89.42% in polyprotein 1 and 73.61%-85.78% in polyprotein 2). A comparative analysis based on taxonomic criteria for the species demarcation of nepoviruses confirmed that they are not new species but distinct variants. This is the first report of the complete genome sequences of CNSV detected in P. suffruticosa and D. odora, and the first report of CNSV infecting P. suffruticosa.


Assuntos
Daphne/virologia , Genoma Viral , Nepovirus/classificação , Nepovirus/isolamento & purificação , Paeonia/virologia , Análise de Sequência de DNA , Nepovirus/genética , Filogenia , Poliproteínas/genética , RNA Viral/genética , Homologia de Sequência de Aminoácidos
16.
J Virol Methods ; 271: 113673, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31170470

RESUMO

Pollen transmitted viruses require accurate detection and identification to minimize the risk of spread through the global import and export of pollen. Therefore in this study we developed RT-qPCR assays for the detection of Cherry leaf roll virus (CLRV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry virus A (CVA), four viruses that infect pollen of Prunus species. Assays were designed against alignments of extant sequences, optimized, and specificity was tested against known positive, negative, and non-target controls. An examination of assay sensitivity showed that detection of virus at concentrations as low as 101 copies was possible, although 102 copies was more consistent. Furthermore, comparison against extant assays showed that in both pollen and plant samples, the newly developed RT-qPCR assays were more sensitive and could detect a greater range of isolates than extant endpoint RT-PCR and ELISA assays. Use of updated assays will improve biosecurity protocols as well as the study of viruses infecting pollen.


Assuntos
Abastecimento de Alimentos , Vírus de Plantas/genética , Vírus de Plantas/isolamento & purificação , Pólen/virologia , Prunus/virologia , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Flexiviridae/genética , Flexiviridae/isolamento & purificação , Ilarvirus/genética , Ilarvirus/isolamento & purificação , Nepovirus/genética , Nepovirus/isolamento & purificação , Doenças das Plantas/virologia , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Análise de Sequência de DNA
17.
Virol J ; 16(1): 70, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133023

RESUMO

A novel nepovirus was identified and characterised from caraway, and tentatively named caraway yellows virus (CawYV). Tubular structures with isomeric virus particles typical for nepoviruses were observed in infected tissues by electron microscopy. The whole genome of CawYV was identified by high throughput sequencing (HTS). It consists of two segments with 8026 nt for RNA1 and 6405 nt for RNA2, excluding the poly(A) tails. CawYV-RNA1 shared closest nt identity to peach rosette mosaic virus (PRMV) with 63%, while RNA2 shared 41.5% with blueberry latent spherical virus (BLSV). The amino acid sequences of the CawYV protease-polymerase (Pro-Pol) and capsid protein (CP) regions share the highest identities with those of the subgroup C nepoviruses. The Pro-Pol region shared highest aa identity with PRMV (80.1%), while the CP region shared 39.6% to soybean latent spherical virus. Phylogenetic analysis of the CawYV-Pro-Pol and -CP aa sequences provided additional evidence of their association with nepoviruses subgroup C. Based on particle morphology, genomic organization and phylogenetic analyses, we propose CawYV as a novel species within the genus Nepovirus subgroup C.


Assuntos
Carum/virologia , Nepovirus/classificação , Doenças das Plantas/virologia , Folhas de Planta/virologia , Proteínas Virais/genética , Proteínas do Capsídeo/genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Nepovirus/isolamento & purificação , Filogenia , RNA Viral/genética , Homologia de Sequência de Aminoácidos
18.
Sci Rep ; 9(1): 7313, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086246

RESUMO

The soil-borne nematode Xiphinema index is closely linked to its main host, the grapevine, and presents a major threat to vineyards worldwide due to its ability to transmit Grapevine fanleaf virus (GFLV). The phylogeography of X. index has been studied using mitochondrial and microsatellite markers in samples from most regions of its worldwide distribution to reveal its genetic diversity. We first used the mitochondrial marker CytB and illustrated the low intraspecific divergence of this mainly meiotic parthenogenetic species. To generate a higher polymorphism level, we then concatenated the sequences of CytB and three mitochondrial markers, ATP6, CO1 and ND4, to obtain a 3044-bp fragment. We differentiated two clades, which each contained two well-supported subclades. Samples from the eastern Mediterranean and the Near and Middle East were grouped into three of these subclades, whereas the samples from the western Mediterranean, Europe and the Americas all belonged to the fourth subclade. The highest polymorphism level was found in the samples of one of the Middle and Near East subclades, strongly suggesting that this region contained the native area of the nematode. An east-to-west nematode dissemination hypothesis appeared to match the routes of the domesticated grapevine during Antiquity, presumably mainly dispersed by the Greeks and the Romans. Surprisingly, the samples of the western subclade comprised only two highly similar mitochondrial haplotypes. The first haplotype, from southern Iberian Peninsula, Bordeaux and Provence vineyards, exhibited a high microsatellite polymorphism level that suggests introductions dating from Antiquity. The second haplotype contained a highly predominant microsatellite genotype widespread in distant western countries that may be a consequence of the massive grapevine replanting following the 19th-century phylloxera crisis. Finally, our study enabled us to draw a first scaffold of X. index diversity at the global scale.


Assuntos
Vetores de Doenças , Nematoides/genética , Nepovirus/isolamento & purificação , Doenças das Plantas/virologia , Vitis/virologia , Animais , DNA de Helmintos/genética , DNA de Helmintos/isolamento & purificação , Domesticação , Genes de Helmintos/genética , Marcadores Genéticos/genética , Haplótipos , Espécies Introduzidas , Região do Mediterrâneo , Repetições de Microssatélites/genética , Oriente Médio , Nematoides/virologia , Filogenia , Filogeografia , Polimorfismo Genético , Solo , Vitis/parasitologia
19.
Mol Plant Microbe Interact ; 32(7): 790-801, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30640575

RESUMO

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.


Assuntos
Nepovirus , RNA Viral , RNA Polimerase Dependente de RNA , Tabaco , Mutação , Nepovirus/enzimologia , Nepovirus/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Tabaco/virologia
20.
Virology ; 524: 127-139, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30195250

RESUMO

ARGONAUTEs (notably AGO1 and AGO2) are effectors of plant antiviral RNA silencing. AGO1 was shown to be required for the temperature-dependent symptom recovery of Nicotiana benthamiana plants infected with tomato ringspot virus (isolate ToRSV-Rasp1) at 27 °C. In this study, we show that symptom recovery from isolate ToRSV-GYV shares similar hallmarks of antiviral RNA silencing but occurs at a wider range of temperatures (21-27 °C). At 21 °C, an early spike in AGO2 mRNAs accumulation was observed in plants infected with either ToRSV-Rasp1 or ToRSV-GYV but the AGO2 protein was only consistently detected in ToRSV-GYV infected plants. Symptom recovery from ToRSV-GYV at 21 °C was not prevented in an ago2 mutant or by silencing of AGO1 or AGO2. We conclude that other factors (possibly other AGOs) contribute to symptom recovery under these conditions. The results also highlight distinct expression patterns of AGO2 in response to ToRSV isolates and environmental conditions.


Assuntos
Antivirais/metabolismo , Proteínas Argonauta/metabolismo , Interações Hospedeiro-Patógeno , Nepovirus/patogenicidade , Doenças das Plantas/virologia , Tabaco/virologia , Proteínas Argonauta/genética , Resistência à Doença , Nepovirus/genética , Doenças das Plantas/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Interferência de RNA , Tabaco/imunologia , Virulência
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