Monoclonal antibodies for the detection and differentiation of faba bean necrotic yellows virus isolates

Abstract

Murine monoclonal antibodies (MAbs) were produced for the detection of faba bean necrotic yellows virus (FBNYV), an isometric ssDNA virus belonging to a new, yet unnamed genus of plant viruses. A total of 19 FBNYV-specific MAbs were obtained from three fusion experiments and characterised by determining their immunoglobulin types and titres as well as their corresponding epitopes. At least six distinct epitopes were revealed on FBNYV particles of different virus isolates. Only two MAbs reacted with SDS-dissociated FBNYV virions in triple antibody sandwich (TAS)-ELISA and with viral capsid protein in Western blots. Almost all MAbs were more sensitive in detecting FBNYV in viruliferous aphids by TAS-ELISA than polyclonal anti-FBNYV IgG by double antibody sandwich ELISA and permitted virus detection in individual aphids even following short acquisition access feeding periods.Coat protein variation among FBNYV isolates and serological relatedness to taxonomically similar viruses was studied by determining the cross reactivity of these MAbs with several field isolates of FBNYV as well as with milk vetch dwarf (MDV), banana bunchy top (BBTV), and subterranean clover stunt (SCSV) viruses. Whereas none of the MAbs reacted with BBTV, only one reacted with SCSV, indicating that FBNYV and SCSV share a common epitope. By contrast, 16 of the 19 MAbs reacted with MDV, suggesting that FBNYV and MDV are serologically closely related and strains of the same virus. When all 19 MAbs produced were tested against a total of 107 samples of FBNYV collected during virus surveys in Egypt, Ethiopia, Jordan, Morocco and Syria, five MAbs showed differential reactions. While the majority of the samples reacted with all 19 MAbs, about 20% of the 107 FBNYV samples did not react with one and/or other of these live MAbs, permitting the differentiation of seven serotypes of FBNYV and suggesting a considerable coat protein variation in FBNYV isolates from the countries surveyed. The MDV isolate from Japan and five FBNYV samples from Ethiopia appeared to be the least closely related to typical FBNYV isolates by not reacting with three and four, respectively, of the five differentiating Mabs.

Reconstitution of Authentic Nanovirus from Multiple Cloned DNAs

“…Moreover, FBNSV-[ET:Hol:97] failed to react with the FBNYV MAbs 1-3D8, 2-5H9, and 3-4A5. For instance, MAbs 2-5H9 and 3-4A5 reacted in TAS-ELISA with FBNYV but not with MDV and a nanovirus in faba bean samples originating from Ambo, Ethiopia (18). This suggested that FBNSV-[ET:Hol:97] is serologically distinct from Syrian and Egyptian isolates of FBNYV as well as from MDV.…”

“…Each of two female BALB/c mice (2 to 3 months old) received a subcutaneous injection of ϳ75 g of FBNSV virions in 150 l containing 50% Freund's incomplete adjuvant. Booster injections, fusion experiments, selection of hybridomas, and cloning were essentially conducted as described previously (1,18). For hybridoma selection by TAS-ELISA, however, FBNYV IgG was used for the trapping of antigen from extracts of FBNYV-and FBNSV-infected faba bean plants as well as from noninoculated plants.…”

“…For serological analysis of inoculated plants, triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) was performed as described previously (18), using highbinding polystyrene plates (Greiner Bio-One GmbH, Germany), the buffers described by Clark and Adams (12), and the nanovirus-specific monoclonal antibodies (MAbs) 1-1F2, 2-1A1, and 3-4F2 (18) and the broad-spectrum luteovirid-specific MAb B-2-5G4 (29) as detecting antibodies. For trapping of nanovirus and luteovirid antigen in TAS-ELISA, we coated plates with immunoglobulin G (IgG) to FBNYV and turnip yellows virus, respectively.…”

Ten distinct circular ssDNA components, four of which encode putative replication-associated proteins, are associated with the faba bean necrotic yellows virus genome.

“…A serological study with a range of monoclonal antibodies to FBNYV showed that an MDV isolate from Japan shared four of the six identified FBNYV epitopes with typical FBNYV isolates from Syria and Egypt, suggesting that FBNYV and MDV might be strains of the same virus (Franz et al, 1996). Although FBNYV and MDV are most similar in one of their non-Rep proteins (90n8 % identity) and have two apparently closely related Rep proteins (88n3 and 89n8 % identical), their coat protein amino acid sequences were 83n6 % identical (Sano et al, 1998), and they differed in their four other non-Rep proteins by 23 %.…”

Breeding for biotic stress resistance in chickpea: progress and prospects

“…It is the causal agent of a destructive disease of chickpea in some arid regions, including countries in north Africa, west Asia and southern Europe (Katul et al 1993;Franz et al 1996). FBNYV is known to infect more than 50 legume species.…”