Detection of four regulated grapevine viruses in a qualitative, single tube real-time PCR with melting curve analysis.

The detection of the four grapevine viruses (GLRaV-1, GLRaV-3, GFLV and ArMV) regulated in European Union plant material certification, requires sensitive and specific diagnostic tools. A strategy of simultaneous detection in a real-time single tube amplification was developed, based on the EvaGreen binding dye. The melting curve analysis (MCA) of the amplicons allows a qualitative detection of the four different virus targets in multiplex analysis. A plasmid dilution assay calculated an analytical sensitivity with an amplification threshold up to 100 copies of the target sequences. A small cohort of field grapevine samples, with a known status of infection by mixtures of the target viruses or free of them, respectively, was successfully tested for the evaluation of the amplicons Tm.

Discovery and molecular characterization of a new cryptovirus dsRNA genome from Japanese persimmon through conventional cloning and high-throughput sequencing.

Through the application of next generation sequencing, in synergy with conventional cloning of DOP-PCR fragments, two double-stranded RNA (dsRNA) molecules of about 1.5 kbp in size were isolated from leaf tissue of a Japanese persimmon (accession SSPI) from Apulia (southern Italy) showing veinlets necrosis. High-throughput sequencing allowed whole genome sequence assembly, yielding a 1,577 and a 1,491 bp contigs identified as dsRNA-1 and dsRNA-2 of a previously undescribed virus, provisionally named as Persimmon cryptic virus (PeCV). In silico analysis showed that both dsRNA fragments were monocistronic and comprised the RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP) genes, respectively. Phylogenetic reconstruction revealed a close relationship of these dsRNAs with those of cryptoviruses described in woody and herbaceous hosts, recently gathered in genus Deltapartitivirus. Virus-specific primers for RT-PCR, designed in the CP cistron, detected viral RNAs also in symptomless persimmon trees sampled from the same geographical area of SSPI, thus proving that PeCV infection may be fairly common and presumably latent.

Identification and characterization of a viroid resembling apple dimple fruit viroid in fig (Ficus carica L.) by next generation sequencing of small RNAs.

Viroids are small (246-401 nt) circular and non coding RNAs infecting higher plants. They are targeted by host Dicer-like enzymes (DCLs) that generate small RNAs of 21-24 nt (sRNAs), which are involved in the host RNA silencing pathways. The accumulation in plant tissues of such viroid-derived small RNAs (vd-sRNAs) is a clear sign of an ongoing viroid infection. In this study, next generation sequencing of a sRNAs library and assembling of the sequenced vd-sRNAs were instrumental for the identification of a viroid resembling apple dimple fruit viroid (ADFVd) in a fig accession. After confirming by molecular methods the presence of this viroid in the fig tree, its population was characterized, showing that the ADFVd master sequence from fig diverges from that of the ADFVd reference variant from apple. Moreover, since this viroid accumulates at a low level in fig, a semi-nested RT-PCR assay was developed for detecting it in other fig accessions. ADFVd seems to have a wider host range than thought before and this poses questions about its epidemiology. A further characterization of ADFVd-sRNAs showed similar accumulation of (+) or (-) vd-sRNAs that mapped on the viroid genome generating hotspot profiles. Moreover, similarly to other nuclear-replicating viroids, vd-sRNAs of 21, 22 and 24 nt in size prevailed in the distribution profiles. Altogether, these data support the involvement of double-stranded RNAs and different DCLs, targeting the same restricted viroid regions, in the genesis of ADFVd-sRNAs.

Production of antibodies to Little cherry virus 1 coat protein by DNA prime and protein boost immunization.

Little cherry, an economically important disease of cherry is caused by at least two different viruses. One of these is Little cherry virus 1 (LChV-1) for the detection of which no efficient serological tools are available, so that diagnosis is based on molecular methods. In this study, different immunization strategies for producing antibodies against the viral coat protein of LChV-1 were tried, using either purified virus preparations, or bacterially expressed protein, or a DNA vector that expressed the cloned coat protein (CP) gene in vivo. Effective induction of specific antibodies to LChV-1 CP was obtained using DNA intramuscular immunization followed by a single boost with the recombinant protein. The entire coat protein sequence was cloned in a mammalian expression vector and, after being coated by an amphiphilic non-toxic reagent was delivered into rabbit. A protein boost increased the specific immune response against the virus protein. The sensitivity of this antiserum is lower if compared with that of antisera raised conventionally against other viruses, thus it requires improvements for use for diagnostic purposes.

Identification of an RNA-silencing suppressor in the genome of Grapevine virus A.

Higher plants use post-transcriptional gene silencing (PTGS), an RNA-degradation system, as a defence mechanism against viral infections. To counteract this, plant viruses encode and express PTGS suppressor proteins. Four of the five proteins encoded by the Grapevine virus A (GVA) genome were screened using a green fluorescent protein (GFP)-based transient expression assay, and the expression product of ORF5 (protein p10) was identified as a suppressor of silencing. ORF5 p10 suppressed local and systemic silencing induced by a transiently expressed single-stranded sense RNA. This protein was active towards both a transgene and exogenous GFP mRNAs. Ectopic expression of GVA-ORF5 by a Potato virus X vector enhanced symptom severity. The findings that p10 markedly reduces the levels of small interfering RNAs (siRNAs) and that the recombinant protein is able to bind single-stranded and double-stranded forms of siRNAs and microRNAs, suggest the existence of a potential mechanism of suppression based on RNA sequestering.

Isolation of recombinant antibodies (scFvs) to grapevine virus B.

A panel of 15 recombinant single chain antibodies (scFv) specific to grapevine virus B (GVB) were recovered from a human combinatorial scFv antibody library using the phage display technique against purified virus particles. Two selected scFv-encoding genes were expressed in recombinant Escherichia coli cells as dimeric antibodies. Successful detection of GVB in tissues of herbaceous hosts and grapevine was obtained in a direct binding assay using dimeric scFvs. This reagent was also shown to substitute efficiently for a GVB polyclonal serum in standard DAS-ELISA test used routinely for diagnosis.

Epitope mapping of Grapevine virus A capsid protein.

Previously characterized monoclonal antibodies (MAbs) to Grapevine virus A (GVA) showed a differential reactivity against intact or partially destabilized virus particles [2]. In the present study, this differential reactivity was confirmed and several peptides reacting with a panel of four different antibodies were identified by the PEPSCAN method of epitope mapping. Oligopeptide sequences comprised between coat protein residues 61 (V) and 72 (T) were recognized by all the antibodies tested. One of these peptides (VGPKASK) was also reactive when expressed on recombinant phage particles as a fusion protein with protein pVIII. The specificity of this sequence for antibody binding was also demonstrated by competitive-ELISA using one of the GVA MAbs. The results of this study suggest that GVA particles carry a highly structured epitope centered on a common peptide region of the coat protein sequence.

Immunodetection and subcellular localization of the proteins encoded by ORF 3 of grapevine viruses A and B.

Polyclonal antisera were raised to Escherichia coli-expressed ORF3 products (putative movement proteins) of Grapevine virus A (GVA) and Grapevine virus B (GVB) (genus Vitivirus), and used for their immunodetection in infected plants. Western blot analysis of subfractionated cellular compartments showed that the distribution of both proteins was comparable to that of plant virus movement proteins, as they were transiently present in a crude membrane fraction and accumulated in a cell wall-enriched fraction. The GVA ORF3-encoded protein, but not the comparable GVB protein, was also present in large amount in a cytoplasmic soluble fraction. Intracellular immunogold labelling localized these proteins in the cell wall and plasmodesmata of infected cells and, especially for GVA, in association with cytoplasmic virus aggregates.

Infectious cDNA clones of two grapevine viruses.

Full-length cDNA copies of the genomes of Grapevine virus A (GVA) and Grapevine virus B (GVB) under the control of bacteriophage T7 promoter have been synthesized, which were refractory to cloning in Escherichia coli. However, both transcribed cDNAs were infectious when mechanically inoculated to Nicotiana plants. A full-length cDNA copy of GVB was engineered in pCass2, a plasmid containing a partially duplicated copy of the Ca35S promoter, but was rather unstable in Escherichia coli. No infection of Nicotiana plants was obtained following mechanical inoculation but detached Nicotiana leaves, inoculated by particle bombardment, supported the multiplication of a GVB isolate seemingly identical to the wild-type used for cloning. Nicotiana seedling inoculated with sap expressed from these leaves became infected showing typical GVB symptoms. Transient transcription of Ca35S driven cDNA clones was also detected by RT-PCR in leaves of the grapevine hybrid LN33 following inoculation by particle bombardment. The availability of infectious cDNA clones of GVA and GVB constitutes a tool for the study of genome expression and pathogenesis, and for the ultimate establishment of the aetiological role of these viruses.

A spot-PCR technique for the detection of phloem-limited grapevine viruses.

Specific amplification of genomic fragments of grapevine trichovirus A (GVA), grapevine trichovirus B (GVB) and grapevine leafroll-associated closterovirus 3 (GLRaV3) was obtained by reverse transcription-PCR on total nucleic acid solubilized from small pieces of charged nylon membrane, on which a drop of crude infected grapevine sap was spotted (spot-PCR). A thermal treatment (95 degrees for 10 degrees) of spotted sap in a buffered solution improved the release of viral template. Consistent amplification was obtained with three viruses from fragments of the same respective blots up to 1 month after spotting, while a detection threshold limit comparable with standard PCR techniques was found for this method. Duplex PCR (i.e. amplification of different viruses from a mixed-infected grapevine source) was also found to be effective, since GVA and GLRaV3 were amplified by a mixture of specific primers in the same reaction. This rapid and easy sampling technique, using leaf petioles to express crude sap, may have a wide field application for screening grapevine viruses.

Grapevine virus A: nucleotide sequence, genome organization, and relationship in the Trichovirus genus.

The 5' terminal region of the genomic RNA of grapevine virus A (GVA), a tentative member of the Trichovirus genus, encompassing 5466 nucleotides, was sequenced. Evidence was obtained that the RNA is capped. Two putative open reading frames (ORF) were identified: ORF 1 that codes for a 194 kDa polypeptide with conserved motifs of replication-related proteins of positive-strand RNA viruses, and ORF 2 that encodes a 19 kDa polypeptide with no significant homology with protein sequences from databases. This polypeptide, however, showed 44% similarity with the product expressed by a comparable ORF present in grapevine virus B (GVB). GVA genome had the same size and structural organization as that of GVB. It also had the same size of the genome of apple chlorotic leaf spot virus (ACLSV), the type species of the Trichovirus genus, but differed substantially in the number (5 versus 3), size, and order of genes. Differences existed also in the degree of sequence homology between polymerases, which did not cluster together in phylogenetic trees. Definitive (ACLSV, PVT) and tentative (GVA, GVB) trichovirus species differ molecularly, biologically and epidemiologically to an extent that warrants the taxonomic revision of the genus.

The nucleotide sequence and genomic organization of grapevine virus B.

Grapevine virus B (GVB) is a tentative member of the genus Trichovirus. The 5'-terminal region of the RNA genome of GVB comprises 5437 nucleotides and has been sequenced by the dideoxynucleotide chain termination method. Evidence was obtained that the RNA is capped. Two putative open reading frames (ORFs) were identified. ORF 1 coded for a 194.7 kDa polypeptide with conserved motifs of replication-related proteins of positive-strand RNA viruses (i.e. methyltransferase, helicase and RNA-dependent RNA polymerase, in that order from the N to the C terminus). ORF 2 encoded a 20 kDa polypeptide that did not show any significant sequence homology with protein sequences from the databases. The biological function of this polypeptide was not determined. Although the 20 kDa product was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and an antiserum produced, it could not be identified in GVB-infected plant tissue extracts. The GVB genome had the same size as that of apple chlorotic leaf spot virus (ACLSV), the type species of the genus Trichovirus, but differed substantially in the number (five compared to three), size and order of genes. Differences existed also in the extent of sequence homology between polymerases, which did not cluster together in tentative phylogenetic trees. The results of this study show that definitive and tentative trichovirus species differ molecularly to an extent that may warrant a taxonomic revision of the genus.

Sensitive detection of grapevine virus A, B, or leafroll-associated III from viruliferous mealybugs and infected tissue by cDNA amplification.

DNA primers specific for grapevine virus A (GVA), grapevine virus B (GVB) or grapevine leafroll-associated virus III (GLRaV-III) were constructed based on the nucleotide sequence of a segment of each viral genome. DNA primers were utilized for cDNA synthesis and polymerase chain reaction (PCR) amplification of a 430 bp fragment from extracts of GVA-infected grapevine tissue or viruliferous mealybugs and 450 bp and 340 bp DNA fragments from extracts of GVB and GLRaV-III-infected grapevine tissues, respectively. The amplified DNA fragment of each virus was identified by Southern hybridization analysis with a cRNA probe of cloned viral genome. Reverse transcription (RT)-PCR, immunocapture (IC)-RT-PCR and/or multiplex (M)-RT-PCR assays were developed for the detection of GVA, GVB, and/or GLRaV-III in extracts of infected grapevine leaves, dormant cuttings and/or in viruliferous mealybugs. Viral specific DNA was absent from amplified extracts of uninfected grapevine tissue or nonviruliferious mealybugs. IC-RT-PCR was easier to perform than RT-PCR for the detection of GVA from viruliferous mealybugs. M-RT-PCR was easier and faster than IC-RT-PCR for the detection of GLRaV-III from infected grapevine tissue and it allows the sensitive detection of GVB, for which a high titer antiserum is not yet available.

Nucleotide sequence of the 3' terminal region of the RNA of two filamentous grapevine viruses.

The 3' terminal region of grapevine virus A (GVA) and grapevine virus B (GVB), encompassing 1883 and 2136 nucleotides, respectively, was sequenced by the deoxynucleotide chain termination method. Three putative open reading frames (ORF) were identified in both genomic viral RNAs, denoted 1 to 3 in the 5' to 3' direction. ORF 1 encoded a polypeptide with estimated M(r) of 31 kDa (GVA) and 36.5 kDa (GVB), possessing the G/D motif of the "30 K superfamily" movement proteins, and showing good alignments with putative movement proteins of trichoviruses and capilloviruses. ORF 2 was identified as the coat protein (CP) cistron, coding for polypeptides with an estimated M(r) of 21.5 kDa (GVA) and 21.6 kDa (GVB). These CPs showed substantial sequence homology with one another and with CPs of tricho- and capilloviruses, but not of closteroviruses. ORF 3 potentially coded for two small polypeptides with estimated M(r) of 10 kDa (GVA) and 14 kDa (GVB). The ORF 3 product of GVB (14 K), but not that of GVA, shared some homology with the 3' terminal polypeptides of different plant viruses that exhibit the "zinc finger domain" of proteins with nucleic acid-binding properties. GVA and GVB have many properties in common with trichoviruses but possess an extra open reading frame (ORF 3). Whether this finding may have a bearing on the classification of these viruses is unclear. However, until the taxonomic significance of this difference in genome structure is established, it seems plausible to include GVA and GVB as tentative species in the Trichovirus genus.