Adenylate cyclase (AC) toxin from Bordetella pertussisis a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.

In order to determine if the infectious pancreatic necrosis virus isolate IPNV-Jasper (Ja-ATCC) is homogeneous or heterogeneous with respect to inhibition by normal rainbow trout serum (RTS), 50 clones were tested for sensitivity to RTS. The initial isolate was very sensitive to RTS, losing from 10 4 to 10 8 50% tissue culture infection dose (TCID 50 ) ml -1 with a 1:100 dilution of RTS. The sensitivity of the clones ranged from highly sensitive to completely resistant (0 to 10 8 TCID 50 ml -1 reduction). Eight percent of clones (4/50) were very sensitive to RTS (Ja-S) and 84% of clones (42/50) showed a mid-range of sensitivity to RTS. The final 8% of clones (4/50) were resistant to RTS (Ja-R). Enzyme immunodot assay revealed that Ja-S clones showed a monoclonal reaction identical to the parents, Ja-ATCC; however, Ja-R clones differed by several epitopes from the parental strain. Analysis of Ja-S and Ja-R revealed that there were significant differences in their nucleic acid sequences for the capsid protein VP2. These 2 strains shared 80.7 and 86.5% identity in nucleic acid and in amino acid sequences, respectively. Ja-S had 99.7 and 91.0% identity in nucleic acid sequences, and 99.5 and 95.9% in amino acid sequences with Ja-ATCC and Jasper-Dobos (Ja-D), respectively, while Ja-R showed 80.6 and 79.8% identity in nucleic acid sequences and 86.5 and 87.0% in amino acid sequences with Ja-ATCC and Ja-D, respectively. In conclusion, the Ja-ATCC population was heterogeneous in terms of RTS sensitivity, serotype and cDNA sequences from the VP2 coding region. KEY WORDS: IPNV-Jasper variants · RTS inhibition · Serotype · cDNA sequenceResale or republication not permitted without written consent of the publisher