and 26.6 % for regions I, II, III and IV, respectively (Diallo et al., 1994). In rescue experiments involving morbillivirus or pneumo- virus mini-genomes, only homologous sets of helper plas- mids derived from one virus are able to generate efficient reporter gene expression from heterologous mini-genome constructs (Brown et al., 2005; Stokes et al., 2003). Mixed sets of helper plasmids function only in certain combina- tions, whilst others are inactive or give only a very low level of activity. In fact, substitution of a homologous N helper plasmid with a heterologous N helper plasmid leads to abolition of the reporter gene activity in MV (Brown et al., 2005), RPV (Brown et al., 2005) and the pneumovirus respiratory syncytial virus (Stokes et al., 2003). The incom- patibility of heterologous combinations of helper plasmids to rescue mini-genome reporter genes or full-length gen- omic constructs in paramyxoviruses and pneumoviruses (Brown et al., 2005; Govindarajan et al., 2006) is not unexpected considering, as indicated above, the diverse functions of the N protein. Therefore, it was remarkable that substitution of the N gene in the RPV chimera was successful and fully functional, indicating that the N protein of RPV is replaceable with that of the related PPRV. In contrast, the H protein gene of RPV cannot be replaced with that of PPRV; virus can be rescued only when both the H and F genes are substituted together (Das et al., 2000). In addition, it requires the presence of the PPRV M protein gene for efficient growth in cell culture (Mahapatra et al., 2006). The PPRV N gene has been reported to be 68.8 % identical at the nucleotide level and 72.9 % similar at the amino acid level to the RPV N gene, the highest value when compared with other morbilliviruses (Diallo et al., 1994), and therefore the success of recovering virus from a RPV cDNA clone where the N protein gene has been replaced with that from PPRV may be partly attributable to this high degree of sequence conservation. The chimeric virus reported here had an initial slow growth, which was compensated following further incubation, and it grew to a final titre as high as the parental RPV, as has been observed in a recombinant NDV where an immunodominant epitope in the N protein was deleted (Mebatsion et al., 2002). The only other report showing the exchange of N proteins between two related paramyxoviruses is by Bailly et al. (2000). They recovered a viable recombinant human para- influenzavirus 3 (HPIV3) where the N gene ORF was replaced with that from bovine parainfluenzavirus 3 (BPIV3) and the recombinant virus also grew to a titre as high as that of the parental HPIV3 and BPIV3. A global rinderpest eradication programme is currently under way with a target date of 2010 to achieve this goal. As a result of this effort, rinderpest has been eliminated from Asia and is now confined to southern Somalia in eastern Africa. All countries, including Somalia, have now ceased vaccination and have started intensive disease surveillance and serosurveillance to detect rinderpest anti- bodies in non-vaccinated animals. Part of the final strategy for eradication of rinderpest is an emergency vaccination http://vir.sgmjournals.org Chimeric rinderpest virus plan to prevent spread of the virus in the event of isolated outbreaks of disease in the endemic region. For this eventuality, it is desirable to have a ‘marker vaccine’, which would enable identification of the vaccinated animals serologically and so not interfere with the ongoing sero- surveillance work in other regions of the African continent. Using reverse genetics techniques, positively marked vaccines have been produced for rinderpest (Walsh et al., 2000a, b). These vaccines are highly effective in protecting animals from virulent rinderpest challenge. However, if these vaccines are used, it will not be possible to identify vaccinated animals that subsequently become infected, and this could then mask the presence of the wild-type virus. We have now produced a negative marker vaccine by replacing the N protein gene of RPV with the equivalent gene from PPRV. This vaccine does not produce a strong N protein-specific antibody response as found in natural infection or in traditional vaccination. A preliminary vaccination trial was conducted to evaluate the chimeric RPV–PPRN virus as a marker vaccine and also to compare its efficacy with the tissue-culture- attenuated Plowright rinderpest vaccine. The absence of clinical response, including fever and leukopenia, showed that the vaccine is safe to use, at least in cattle. Moreover, the absence of viral RNA in ocular secretions throughout the period of study, as evidenced by RT-PCR, further confirmed its safety. Neutralizing antibodies were detected in all vaccinated animals, and animals vaccinated with the chimeric vaccine exhibited comparable neutralizing anti- body titres to the RPV-vaccinated animals, indicating the chimeric virus to be as immunogenic as the parental RPV. All of the animals were protected against virulent virus challenge, indicating that it is an efficacious vaccine. The cELISA based on responses to the H protein showed that all of the vaccinated animals were positive for RPV-specific anti-H antibodies, whereas they remained negative for PPRV H-specific inhibition at 6 weeks following vaccina- tion. However, the existing N protein-based cELISAs to detect antibodies against the RPV and PPRV N proteins were found to cross-react with each other and therefore were not suitable for use as serological tests with this new chimeric vaccine. Our results confirmed those reported previously on cross-reactivity between the two tests (Diallo, 2000). In order to circumvent this problem, we expressed the RPVNv protein in E. coli and used it in an indirect ELISA. The new RPVNv ELISA clearly did not show cross- reaction with PPRV N antibodies and is therefore suitable for use as a companion diagnostic test with this vaccine. The lack of response to RPVNv following challenge indi- cated that the vaccine provided sterile immunity to rinder- pest, at least in the short term, as for the conventional Plowright tissue culture vaccine. As the RPV–PPRN vaccine lacks the highly antigenic RPV N protein, it can be used as a marker RPV vaccine to distinguish serologically between vaccinated and naturally recovered animals. The animals vaccinated with the chimeric vaccine will be positive in RPV H cELISA and negative in RPVNv ELISA, whereas the 2025