S. Parida and others The N proteins of negative-stranded RNA viruses are highly antigenic in nature. However, in the case of morbilliviruses, N protein-specific immunity alone does not confer protection (Nakamura et al., 1998; Ohishi et al., 1999). The N protein has been used as an antigen for diagnostic purposes for rabies virus (Katayama et al., 1999), measles virus (MV) (Warnes et al., 1994), vesicular stomatitis virus (Ahmad et al., 1993), Newcastle disease virus (NDV) (Errington et al., 1995) and PPRV (Dechamma et al., 2006; Libeau et al., 1995). Therefore, it appears to be a suitable target for developing a marker vaccine for RPV, as it involves swapping only one of the viral internal protein genes between two virus vaccine strains. The C-terminal variable region of the N protein of morbilliviruses has been reported to protrude from the surface of the viral nucleo- capsid (Heggeness et al., 1981) and is, therefore, a good candidate for developing a test for RPV/PPRV differential diagnosis. The C-terminal variable region of the RPV N protein was expressed in Escherichia coli and used sub- sequently to develop an indirect ELISA that could be used for serological identification of animals vaccinated with the chimeric vaccine. It is proposed that the chimeric virus, when used in conjunction with the newly developed com- panion serological test, would be suitable for use in the final phase of the rinderpest eradication campaign. METHODS Cell culture and viruses. Vero cells (an African green monkey kidney cell line) were grown in Dulbecco’s minimal essential medium containing 25 mM HEPES (pH 7.2) and 5 % fetal calf serum with penicillin (100 IU ml21) and streptomycin (100 mg ml21). Recombinant RPV (RPV2C) (Mahapatra et al., 2006) and the vaccine strain of PPRV (Nigeria 75/1; Diallo et al., 1989) were grown in Vero cells. When the cytopathic effect (CPE) was almost complete (usually 4–5 days for RPV and 8–9 days for PPRV), virus was harvested by a single freeze–thaw cycle of infected cells and, after removal of cell debris by centrifugation at 1280 g for 10 min, aliquots were stored at 270 uC. Both RPV and PPRV were titrated on Vero cells by determination of TCID50. The rescued recombinant virus was grown and titrated on Vero cells as described above. Recombinant fowlpox virus was grown in chicken embryo fibroblasts as previously described (Mahapatra et al., 2006). Plasmids and molecular biology techniques All DNA manipulations and cloning were carried out using standard protocols. The plasmids pKSN, pKS-P, pGEM-L and pRPV2C have been described elsewhere (Baron & Barrett, 2000). Sequencing of DNA was performed using the T7 sequencing kit (Pharmacia Biotech) following the manufacturer’s instructions. RNA extraction from cultures, purified peripheral blood lymphocytes (PBLs) and eye swabs was carried out using Trizol (Invitrogen) as described by Mahapatra et al. (2006). RT-PCR using Taq polymerase for analytical purposes and Pfu polymerase for preparative purposes was performed using standard protocols. (i) Cloning of the N gene of PPRV and construction of full-length genome plasmid. In order to manipulate the N gene, the restriction sites ClaI (at the beginning of the N gene) and PacI (immediately after the ORF of the N gene) of plasmid pRPV2C were used. Thus, the same two restriction sites were incorporated into the PPRV N gene 2020 copy. PPRV N gene sequence data (Diallo et al., 1994) was used to design upstream primer PPRNF (59-GCGCATCGATATGGCTAC- TCTCCTTA-39, nt 22–47 incorporating a ClaI site (underlined) immediately before the N gene ORF and PPRNR (59-CGCG- TTAATTAATGATTTGGACGGAGGGTGCGT-39, nt 1646–1614), in- corporating a PacI restriction site (underlined) at the end of PPRV N gene (25 nt downstream of the stop codon). The N gene ORF was amplified using RNA from Vero cells infected with the PPRV vaccine strain and the 1600 bp amplified product was cloned into the vector pT7Blue. A clone containing the N gene ORF of PPRV (pT7BluePPRN) was identified and completely sequenced on both strands. The sequence was compared with the published sequence to ensure that there were no PCR-induced mutations. The ClaI/PacI digestion product of plasmid pT7BluePPRN was used to replace the N gene ORF of pRPV2C to make the full-length genome plasmid pRPV-PPRN. Restriction enzyme analysis was carried out to confirm that the plasmids contained full-length copies of the viral genome and the plasmid was sequenced to check that the N gene was from PPRV. (ii) Cloning of the C-terminal variable region of the N gene of RPV. The C-terminal variable region of the rinderpest virus N gene (RPVNv) was amplified using the upstream primer RPNF (59- GCGTAGGATCCGAAATGGTAAGAAGGTCAGC-39, nt 1197–1227, BamHI site underlined) and the downstream primer RPNR (59- CTCGTGAAGCTTGTCGTTGTATGC-39, nt 1658–1681, HindIII site underlined) using RNA from Vero cells infected with the RPV vaccine strain (RBOK). The 462 bp amplified product was cloned into pT7Blue and named pRPVNv. A clone was completely sequenced on both the strands and the sequence was compared with the published sequence to ensure that there were no PCR-induced mutations. The BamHI/HindIII digestion product of the plasmid pRPVNv was ligated into a similar site in the bacterial expression vector pQE30 (Qiagen) to produce the plasmid pQE30-RPVNv. This vector adds an N- terminal His tag to the coding sequence of the protein. Expression and purification of His-tagged protein. To express the protein, plasmid pQE30-RPVNv was transformed into E. coli strain M15 (Novagen) and grown in NZYCM medium containing ampicillin (50 mg ml21) and kanamycin (25 mg ml21). Expression was carried out essentially as described in the manufacturer’s proto- col. The protein was tested for its solubility and purified using Ni-NTA resin (Qiagen). The protein samples were analysed by 12 % SDS-PAGE. The concentration of the protein was determined using a protein assay kit (Bio-Rad). Transfection and recovery of infectious recombinant virus. Rescue of the chimeric virus genome was carried out using previously described techniques (Parida et al., 2006). The rescued virus in the transfected cell lysate was amplified by infecting Vero cells and harvesting the virus when extensive CPE was observed. The identity of the recombinant virus was confirmed by RT-PCR followed by sequencing to ensure that the N gene was from PPRV. The plaque morphology of the parental and recombinant viruses was assessed as described by Das et al. (2000). Growth of recombinant virus in tissue culture. Multi-step growth curves were carried out by infecting Vero cells in six-well plates at approximately 70 % confluence with equal m.o.i. of recombinant and the original parental viruses. Virus was allowed to adsorb to the cell monolayers for 1–2 h and unbound virus was removed by washing the cells three times with 2 ml growth medium. Finally, 2 ml growth medium was added to each well and the cells were incubated for different time periods. Each virus growth curve was carried out in duplicate and at each time point (0, 12, 24, 36 and 48 h post- infection), the infected cells were frozen at –70 uC. The virus was Journal of General Virology 88