Comparison and Differentiation of Potyvirus Isolates and Identification of Strain-, Virus-, Subgroup-specific and Potyvirus Group-common Epitopes Using Monoclonal Antibodies

Abstract

A panel of monoclonal antibodies (MAbs) generated against an admixture of 12 potyvirus isolates was used to compare and differentiate diverse potyviruses. Both native and denatured virions of strains of bean yellow mosaic (BYMV), potato virus Y, tobacco etch, pea seed-borne mosaic, iris severe mosaic, iris mild mosaic and asparagus virus-1 potyviruses were used as immunogen and as antigen for screening of the hybridoma cell lines. Thirty cell lines secreting potyvirus-specific antibodies reactive in indirect antigen-coated plate (ACP-) ELISA were selected lbr detailed analysis. All 30 MAbs reacted with at least one strain of BYMV; 11 MAbs reacted with between one and eight of the nine BYMV strains and an additional three MAbs reacted only with isolates within the BYMV subgroup (BYMV, pea mosaic virus and clover yellow vein virus). The remaining 16 MAbs reacted with a BYMV isolate and with at least one of the other 43 potyvirus isolates tested. MAb PTY 1 reacted with all 55 potyvirus isolates tested (representing at least 33 different and distinct aphid-transmissible potyviruses). The potyvirus cross-reactive MAbs generally gave higher reactivity values in ACP-ELISA with dissociated virus than with polyclonal antibody-trapped intact virions in triple antibody sandwich ELISA (i.e. were cryptotopespecific). The BYMV strain-and virus-specific MAbs reacted strongly with both types of antigens (i.e. were metatope-specific). At least 25 distinct epitopes (12 cryptotopes and 13 metatopes) could be identified from the MAb-antigen reactivity patterns. The distribution of these epitopes between virus isolates can be used to detect and differentiate potyviruses in infected plant extracts and to examine virus architectures. Some of these epitopes are shared by potyvirus isolates not previously shown to be serologically related. The broad spectrum-reacting MAb PTY 1 recognizes a cryptotope conserved on all of the aphid-transmissible potyviruses examined and should be a valuable tool for the detection and assay of these potyviruses.

Molecular characterization of a structural epitope that is largely conserved among severe isolates of a plant virus.

“…Similarly, previous studies showed an all-ornone reaction that completely abolished the reactivity because of substitutions of critical residues in the protein (24). Previous studies on mapping epitopes of plant viruses used mutants with single or double amino acid substitutions in the coat protein (25), deletion mutants ofthe coat protein (26,27), and short peptide fragments expressed in E. coli as fusion protein or free protein (28,29). Our results provide direct molecular evidence for the critical role of a single nucleotide alteration and the resulting change in the codon on the reactivity of a monoclonal antibody with severe isolates of a plant virus.…”

Cross-reacting and Heterospecific Monoclonal Antibodies Produced Against Arabis Mosaic Nepovirus

“…The fact that, by using the appropriate screening technique, we were able to identify common epitopes between viruses for which little or no serological relationship has been demonstrated, together with the broad spectrum tests which have been produced recently for potyviruses (Jordan & Hammond, 1991 ;Shukla et al, 1989), and to some extent for luteoviruses (Murphy & D'Arcy, 1991), opens the way for the development of MAbs with a broader spectrum for other virus groups. These will prove to be very useful tools for quarantine testing.…”

A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

“…Nevertheless, certain MAbs that are also raised against native virions such as TEV (44) or JGMV (42) cross-react with other potyviruses. However, mixtures of native and denatured virions of 12 potyvirus isolates have been used for immunization, leading to identification of a broad-spectrum PTY1 MAb from 30 screened MAbs that detects at least 55 potyvirus isolates (52). In contrast, we chose a recombinant protein for use as an immunogen, which was formed by fusion of three conserved core CP regions instead of from virus particles; therefore, we avoided the screening of MAbs that react to species-specific epitopes at the N or C terminus and also increased the opportunity for induction of broad-spectrum MAbs.…”

“…2B and 3B), suggesting that the epitope reacting with this C4 MAb should be sequential and not conformational. Jordan and Hammond have proposed that the epitope type may be determined by I-ELISA and triple antibody sandwich (TAS)-ELISA with disrupted and intact virions, respectively (52). MAbs that react best with plate-trapped, disrupted virus particles and CP subunits, as demonstrated by I-ELISA, recognize antigenic sites as cryptotopes, which are hidden within the intact virion (52).…”