Table 2. Detection of RNA extracted from eye swabs (E) or PBLs by RT-PCR The PCR product obtained using the diagnostic primer set (F3/F4) was used as template for the nested PCR using primer set F3a/F4a. P, Number of animals positive for the presence of viral RNA; T, total number of animals tested; NT, not tested. Data in parentheses indicate values post-challenge. Day Sample RPV–PPRN RPV 0 2 5 7 9 12 14 E PBLs E PBLs E PBLs E PBLs E PBLs E PBLs E PBLs F3/F4 (P/T) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 3/3 (0/3) 0/3 (0/3) 1/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) F3a/F4a (P/T) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) NT (1/3) 0/3 (0/3) 1/2 (1/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) F3/F4 (P/T) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) 2/2 (0/2) 0/2 (0/2) 1/2 (0/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) 0/3 (0/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) F3a/F4a (P/T) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) NT (1/2) 0/2 (0/2) 1/1 (1/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) cut-off value for positive/negative in the tests) of the RPV H-specific mAb binding on the day of challenge (Fig. 3a). Moreover, no appreciable anamnestic response was ob- served following challenge with the virulent Saudi 1/81 strain, suggesting that only low-level replication of the challenge virus took place. None of the animals used in the experiments showed a PPRV H-specific inhibition greater than 22 %, considered non-specific in the test, and it was always lower than the RPV H-specific inhibition (data not shown). Detection of N-specific antibodies using the currently available cELISA cELISA was carried out on serum collected from the experimental animals to measure the RPV and PPRV N- specific antibody response using the existing N protein- based cELISA as described in Methods. All animals vaccinated with either RPV or RPV–PPRN exhibited more than 50 % inhibition of RPV N-specific mAb binding on the day of challenge. Although the values were higher for RPV-vaccinated animals, they were not statistically significant (Fig. 3b). Similarly, all of the animals employed in this study exhibited more than 50 % inhibition of PPRV N-specific mAb binding on the day of challenge. Although RPV–PPRN-vaccinated animals exhibited higher values, again they were not statistically significant (Fig. 3c). These results clearly showed the cross-reaction of PPRV N- specific mAb with RPV N protein and vice versa. http://vir.sgmjournals.org Chimeric rinderpest virus Fig. 3. Antibody response in animals subjected to vaccination and challenge. Serum collected at regular intervals from animals vaccinated with RPV2C (UQ9 and UQ10) or RPV–PPRN virus (UQ11, UQ12 and UQ13) was analysed by cELISA for specific inhibition of RPV H (a), RPV N (b) and PPRV N (c) using mAbs (see Methods for details). The cut-off value between negative and positive results was taken as 50 % inhibition and the mean of duplicate readings is plotted. Arrows indicate the day of challenge. dpv, Days post-vaccination; dpc, days post-challenge. Cloning and expression of the C-terminal variable region of RPV N protein In order to develop a more-specific ELISA, the coding region of the C-terminal variable region of RPV N protein (RPVNv) was amplified by PCR and inserted into the bacterial expression vector pQE30. An N-terminal His- tagged protein was expressed efficiently from this vector in E. coli (Fig. 4a) and found to be soluble in nature. The RPVNv protein was purified on a Ni-NTA spin column to near homogeneity as judged by Coomassie blue staining (data not shown). 2023