S. Parida and others Fig. 4. (a) Expression of RPVNv protein in E. coli. E. coli strain M15 was transformed with the expression plasmid encoding RPVNv protein and grown as described in Methods. Cell lysates were prepared and analysed by 12 % SDS-PAGE. The gel was stained with Coomassie blue and photographed. M, Marker; lane 1, cell lysate from M15 control cells; lanes 2–5, cell lysate from clones 1–4 expressing RPVNv protein. The position of the N protein is indicated by an arrow. (b) 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 indirect ELISA using bacterially expressed RPVNv protein as antigen (see Methods for details). Results are expressed as absorbance values and the mean of duplicate readings is plotted. Arrows indicate the day of challenge. Detection of RPV N-specific antibodies using recombinant RPVNv antigen in an indirect ELISA An indirect ELISA was carried out as described in Methods on serum collected from the vaccinated and challenged animals to detect the antibody response to N protein using the E. coli-expressed RPVNv as the antigen. Animals vac- cinated with RPV–PPRN virus were found to be negative for RPV N-specific antibodies following vaccination. Following challenge with virulent RPV (containing RPV N), the animals remained negative in the RPV N-specific indirect ELISA (Fig. 4b), suggesting that the low level of replication of the challenge virus was not sufficient to elicit a specific antibody response. Animals vaccinated with RPV showed significantly higher levels of RPV N-specific antibodies in comparison with animals vaccinated with RPV–PPRN vaccine on the day of challenge and 1 and 2 weeks following challenge (Fig. 4b). No cross-reaction of the rinderpest antigen (RPVNv) with the PPRV N-elicited antibodies was observed, reflecting the fact that this test 2024 can be used as a diagnostic tool for serological identifica- tion of animals vaccinated with the RPV–PPRN marker vaccine and also for differentiation of RPV- and PPRV- infected animals. Neutralizing antibody titres Serum neutralization assays were carried out to determine the virus neutralizing antibody titres to RPV, if any, in the serum of vaccinated animals before and after challenge with virulent RPV. As shown in Table 3, on day 0, all of the animals were negative for the presence of RPV-specific neutralizing antibody and all of the vaccinated animals had significant neutralizing antibody titres on the day of challenge (ranging from log 3.81 to 4.18). The neutralizing antibody titre of the RPV–PPRN group was found to be as high as that of the RPV group, indicating the chimeric virus to be as immunogenic as the parental RPV. DISCUSSION The paramyxovirus N protein plays an essential role in virus replication by associating with the P and L proteins and is also involved in encapsidation of the viral genome to form the ribonucleocapsid. N–N, N–P, N–P+L and N–M interactions have been reported in paramyxoviruses (Buchholz et al., 1994; Coronel et al., 2001; Curran, 1996; Curran et al., 1998; Ryan & Portner, 1990), indicating the diverse functional nature of the N protein. Alignment of morbillivirus N protein amino acid sequences has defined four regions (I–IV) with varying degrees of sequence homology (Diallo et al., 1994; Griffin & Bellini, 1996). The amino acids in region I (aa 1–122) are well conserved, whereas the residues in region II (aa 123–144) show low sequence homology. Region III (aa 145–420) represents the central highly conserved area, whereas the amino acids in region IV (aa 421–525) at the C terminus are the least conserved. Sequence analysis of PPRV and RPV N proteins revealed amino acid sequence identities of 81.8, 40.9, 90.5 Table 3. Serum neutralizing antibody responses to RPV in cattle vaccinated with RPV–PPRN or RPV Results are given as the log10 titre that gave 50 % neutralization. –, Negative result. Week (post- vaccination) 0 1 2 6 7 8 RPV RPV–PPRN UQ9 UQ10 UQ11 UQ12 UQ13 2 1.45 3.05 3.81 4.26 3.96 2 1.45 2.98 4.18 4.48 4.18 2 1.45 3.13 4.18 4.33 4.11 2 2.2 2.75 4.11 4.41 4.37 2 2.05 2.68 4.11 4.41 4.41 Journal of General Virology 88