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.

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.

Properties of a filamentous virus isolated from grapevines affected by corky bark.

A virus with highly flexuous filamentous particles c. 800 nm long, showing distinct transverse striations was isolated with high frequency (60%) by inoculation of Nicotiana occidentalis with sap from grapevine accessions indexing positive for corky bark. The virus, for which the name grapevine virus B (GVB) is proposed, has an ssRNA genome with mol. wt. of c. 2.5 x 10(6) Da (c. 7600 nt) and coat protein subunits with mol. wt. of c 23,000 Da. GVB has a very restricted herbaceous host range and was experimentally transmitted by the mealybug Pseudococcus ficus. The physicochemical and ultrastructural properties of GVB resemble those of closteroviruses. However, it is serologically unrelated to other grapevine closteroviruses including grapevine virus A, with which it shares some biological and physicochemical properties.

Production, characterization and use of monoclonal antibodies to grapevine virus A.

Four stable hybridoma cell lines secreting monoclonal antibodies to grapevine closterovirus A (GVA) were obtained by fusing spleen cells of immunized BALB/c mice with mouse myeloma cell line Sp2/0-Ag 14. In ELISA all MAbs reacted with virus in leaf extracts from Nicotiana benthamiana, glass-house-, field-, or in vitro-grown grapevines, or with cortical shavings from mature grape canes. In IEM tests, only one of the MAbs (PA3.F5) decorated virus particles on the entire surface. This MAb was likely induced by a surface antigenic determinant, whereas the other three MAbs (PA3.D 11, PA3.C 6, and PA3.B 9) were originated by cryptotopes. Coupling polyclonal antibodies for coating protein A-sensitized plates, and monoclonal antibody conjugates for antigen detection, gave highly efficient and reproducible results for identification of GVA in field-grown grapevines.