S. Parida and others magnitude of replication of RPV–PPRN virus were comparable to those of the parental viruses, indicating that the recombinant virus was fully competent for multi- cycle growth in vitro. This experiment was repeated with similar results. In vivo characterization of RPV–PPRN virus A preliminary vaccination trial was carried out to study the effectiveness of the chimeric virus in generating protective immunity and stimulating an antibody response in the natural host species (cattle) by comparing it with the parental rinderpest vaccine. At 6 weeks post-vaccination, all cattle were challenged with the highly virulent RPV Saudi 1/81 strain. A control group was not included in the experiment, as the challenge virus was from a batch of well- characterized, freeze-dried virus that has been used in previous animal experiments and is known to be highly virulent (Kamata et al., 2001; Walsh et al., 2000b). No specific signs of clinical disease associated with RPV infection were observed in any of the five RPV–PPRN- or RPV-vaccinated cattle following vaccination. The rectal temperature and leukocyte counts of the animals employed in this study were monitored for 2 weeks post-vaccination and challenge as indicators of subclinical disease. The rectal temperature of all of the vaccinated animals remained within the normal range following vaccination (data not shown). In the majority of vaccinated animals, there was an initial leukopenia on day 2, after which levels returned to normal (Fig. 2a). This mild leukopenia is commonly observed in cattle vaccinated with the standard RBOK vaccine and indicates replication of the vaccine virus. Fig. 2. Leukocyte counts in the blood of animals following vaccination (a) and challenge (b). Animals were vaccinated with RPV2C (UQ9 and UQ10) or RPV–PPRN virus (UQ11, UQ12 and UQ13) and the leukocyte count per mm3 of blood was recorded on days 0, 2, 5, 7, 9, 12 and 14 following challenge. 2022 Following challenge, none of the vaccinated animals showed any constant rise in body temperature (data not shown) or exhibited any rinderpest-specific clinical signs. A mild transient leukopenia was observed following challenge in two animals, one from each group (Fig. 2b), suggesting minimal replication of the challenge virus. A much more dramatic and severe leucopenia occurs in animals infected with virulent RPV (Anderson et al., 1996; Walsh et al., 2000b) Detection of virus and viral RNA in clinical samples Attempts were made to isolate virus from the PBLs of vaccinated animals. Virus could be isolated from some animals vaccinated with chimeric virus on days 2 and 5 (Table 1). This is in agreement with our observation of the presence of viral RNA in lymphocytes on this day as evidenced by RT-PCR (Table 2). No viral RNA was detected in occular swabs during the entire period of the study, a characteristic of the parental rinderpest vaccine, which is not spread by contact, indicating it to be a safe vaccine. Viral RNA in lymphocytes was detected mainly on days 2 and 5 following vaccination, after which it began to dis- appear and could only be detected by nested PCR (Table 2). Following challenge, there was evidence of slow replication of challenge virus in some vaccinated animals, which was not sufficient to be detected by virus isolation or a simple diagnostic PCR, but could be detected in some animals by the more sensitive nested PCR on days 2 and 5 (Table 2). Detection of H-specific antibodies cELISA was carried out on serum collected from the vac- cinated and challenged animals to detect the antibody response using RPV and PPRV H-specific mAbs as described in Methods. None of the animals employed in this study showed an antibody response to RPV H protein on the day of vaccination. All animals vaccinated with RPV or RPV–PPRN showed more than 50 % inhibition (the Table 1. Virus isolation PBLs isolated from each animal following vaccination and challenge were co-cultivated with Vero cells. P, Number of animals positive for virus isolation; T, total number of animals tested. Numbers in parentheses are values post-challenge. Day 0 2 5 7 9 12 14 RPV–PPRN (P/T) 0/3 (0/3) 1/3 (0/3) 1/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) 0/3 (0/3) RPV (P/T) 0/2 (0/2) 1/2 (0/2) 1/2 (0/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) 0/2 (0/2) Journal of General Virology 88