1University of Florida, CREC, Lake Alfred, FL 33850, USA; 2USDA-ARS Horticultural Res. Lab., Orlando, FL 32803, USA

Background and objectives
Citrus tristeza virus (CTV), a member of the closterovirus group, has a filamentous, thread-like virion containing a single-stranded positive-sense RNA of 19.3 kb which is encapsidated with multiple copies of a 25-kDa capsid protein (CP) and a 27-kDa coat protein homology at one end of the particle. The virus infects most Citrus spp. and several citrus relatives and is largely confined in the phloem tissue of infected plants. CTV exists as a complex of isolates or strains which produce a variety of symptoms depending on the host or scion/rootstock [1]. Decline and stem-pitting diseases are most important to commercial production. Management strategies for CTV include regulatory methods that are dependent on appropriate diagnostic procedures. Serological procedures, such as ELISA, are easily adapted to large-scale usage and widely used. However, assays with increased sensitivity and specificity are needed. The objective of this study was to develop an improved understanding of the antigenic structure of CTV to provide a better basis for developing new sero-diagnostic procedures. Antibodies against proteins expressed in E. coli to various regions of the CP gene have been developed. Expressed CTV-CP products have also been used as antigens for determining the location and nature of epitopes recognized by monoclonal antibodies (Mabs) specific for CTV. ELISA assays which have improved sensitivity for detection of CTV from infected plants are reported.

Results and discussion
Epitopes recognized by a panel of 30 CTV-specific Mabs produced against different isolates were mapped on the CTV-CP gene by using antigens obtained from expressing CTV-CP gene deletion mutants in E. coli. Analyses were made by Western blotting and ELISA. All Mabs analyzed could be placed into one of five groups (I to V). Four groups have continuous sequential epitopes: Group 1 epitopes are located within aa 215 to 223 of CP, Group II epitopes are between aa 173 to 215, Group III epitopes are between aa 118 to 128, and Group IV epitopes are between aa 2 to 121. Group V are conformational or include a conformation element because they only reacted with virion CP and the complete CP fusion protein, but not with fragments of CP fusion proteins. Proteolytic cleavage of the maltose-binding fusion protein (MBP) also released a full-size CP as well as a MBP, both having no reactivity with Mabs from Group V. The reactivity of different CTV Mabs towards the viral antigen from infected plants, as trapping antibodies or intermediate detecting antibody, was tested using a double antibody sandwich indirect ELISA format. Antibodies to conformational epitopes functioned effectively as trapping antibodies while Group I-IV Mabs were less effective. The trapping function of Groups 1-IV Mabs increased markedly after adding nominal amounts of conformational Mabs, which apparently exposed previously inaccessible cryptic linear epitopes. Similar results were obtained utilizing polyclonal antibodies prepared to fusion proteins of Group I-IV CP regions as trapping antibodies. This modulation of the CTV antigenic reactivity can be used to improve efficiency of trapping antibodies in ELISA-based detection procedures and may also be useful in detecting selected epitopes.

1. Rocha-Pena MA, Lee RF, Lastra R, Niblett CL, Ochoa-Corona FM, Garnsey SM, Yokomi RK, 1995. Plant Disease 79, 437-45.