Differentiation of citrus tristeza virus isolates by serological analysis of p25 coat protein peptide maps

A procedure was developed to purify rapidly and easily a sufficient quantit y of native p25 coat protein (CP) to allow comparison of five isolates of c itrus tristeza virus (CTV) by serological analysis of peptide maps, using m onoclonal and polyclonal antibodies. CTV particles were concentrated by cen trifugation and purified by agarose gel electrophoresis. The CP was extract ed from gel slices riched in virions and protein yields were about three ti mes greater than those obtained previously and of comparable purity. The pu rified CP was partially digested with either V8 or papain endo-protease, an d the peptides generated were separated and electroblotted to a membrane. P rotein blots were tested with four monoclonal antibodies and one source of polyclonal antibodies. The serological maps generated by papain allowed dif ferentiation of all the isolates examined, and those generated by V8 endopr otease allowed discrimination of four of the five isolates tested. Some of these isolates had been indistinguishable based on their reactivity in DASI -ELISA, dsRNA pattern and biological characterization. Serological analysis of peptide maps, as described below, allowed accurate comparison of CTV is olates with minimum amounts of p25 CP and proved superior to other techniqu es for discriminating CTV isolates. (C) 2000 Published by Elsevier Science B.V. All rights reserved.