harvested after one cycle of freeze-thawing and the titre of the released virus was determined by measuring TCID50. Animal studies. Healthy outbred Holstein Freisian bullock calves of approximately 6–12 months of age were used for the vaccination trial, which was carried out in accordance with national legislation governing the use of animals for research and with the approval of the local ethical review committee. Animals were housed in the isolation facility of the Institute for Animal Health and observed for 4 weeks in the isolation unit before the start of the experiment to ensure that they were in good health. Stocks of vaccine virus were grown in Vero cells, and the challenge virus has been described elsewhere (Walsh et al., 2000b). In this experiment, three cows were vaccinated with RPV–PPRN (animal nos UQ11, UQ12 and UQ13) and two with RPV vaccine (animal nos UQ9 and UQ10). Vaccine virus (104 TCID50) was injected into the animals subcutaneously in the shoulder region. All of the animals were challenged with 104 TCID50 virulent RPV (Saudi 1/81) 6 weeks after vaccination. The animals were observed daily for the appearance of clinical signs of rinderpest disease. Rectal temperatures and total leukocyte counts were monitored for 2 weeks following vaccination and challenge. Clinical samples were collected and analysed as described previously (Das et al., 2000). In order to detect the viral RNA in clinical samples (PBLs), a simple diagnostic PCR was carried out using primer set F3 and F4 (Forsyth & Barrett, 1995), specific for the RPV F gene. A nested PCR using the primer set F3a (59-GCTCTGAACGCTATTA- CTAAG-39, nt 845–865) and F4a (59-CTGCTTGTCGTATTTCCTC- AA-39, nt 1079–1059) was also carried out to enhance the sensitivity of detection of the viral RNA; this nested PCR was carried out only on negative samples obtained from the PCR with the diagnostic primer set (F3/F4). Tests for RPV-neutralizing antibodies were carried out in microtitre plates following the method described in OIE (2000). ELISA for detection of H protein-specific antibodies. The virus- specific antibody response was determined using the procedure described by Anderson et al. (1996). This assay determines the amount of antibody in a serum sample that recognizes a specific viral antigen (RPV or PPRV H protein) by the ability of the sample to inhibit binding of an antigen-specific monoclonal antibody (mAb) to viral antigen. The results were expressed as the percentage inhibition of binding of the control mAb. The cut-off value between negative and positive serum was taken as 50 % inhibition. Competitive ELISA (cELISA) for detection of N protein-specific antibodies. cELISA was carried out to measure the antibodies against PPRV N protein using the method described by Libeau et al. (1995). The cELISA carried out to measure the antibodies against RPV N protein was essentially as described by Libeau et al. (1992), except that the antigen used in the ELISA was prepared following the protocol described by Anderson & McKay (1994). Development of an indirect ELISA to detect RPV N-specific antibodies using recombinant RPVNv protein. An indirect ELISA was developed using E. coli-expressed RPVNv protein as the antigen. Plates were coated with RPVNv protein (1 mg ml21) at a 1 : 2000 dilution in carbonate/bicarbonate buffer (pH 9.6) for 1 h at 37 uC. After washing with normal PBS, the serum sample was added at a 1 : 8 dilution in blocking buffer containing 0.1 % Tween 20 and 5 % Marvel in PBS and incubated for a further hour. After a thorough wash, horseradish peroxidase-conjugated anti-bovine IgG (Sigma) was added at a dilution of 1 : 5000 in blocking buffer. After a 1 h incubation at 37 uC, the plates were washed and the colour developed using OPD solution. Colour development was stopped after 10 min by adding 1 M H2SO4. The colour was read in an ELISA plate reader using a 492 mm filter. http://vir.sgmjournals.org Chimeric rinderpest virus RESULTS Recovery of recombinant viruses from cloned cDNA In order to rescue virus from cDNA clones, Vero cells were transfected with the full-length genome plasmid pRPV- PPRN or pRPV2C along with helper N, P and L plasmids. Cells transfected with pRPV-PPRN showed signs of CPE 3–4 days following transfection, at the same time as the control RPV plasmid pRPV2C. Two independent cDNA clones of RPV–PPRN virus were rescued; however, only one was used for further characterization. In order to confirm the identity of the recombinant virus, the N gene was amplified using the PCR primer set PPRNF/PPRNR, which produced an amplified product of the expected size (data not shown). No PCR products were generated in parallel reactions in which the reverse transcriptase was omitted, indicating that the amplified products were not generated from the transfected plasmid DNA. The PCR products were sequenced to ensure that they were from the expected virus. In order to study the plaque morphology of the chimeric virus, Vero cells infected with the parental or chimeric virus were overlaid with carboxy methyl cellulose and the plaques were stained with Giemsa. Phenotypically, RPV–PPRN virus was found to be more or less similar to the parent viruses, i.e. small syncytia were observed (data not shown). Growth of chimeric virus in tissue culture Standard multi-step growth curves for the recombinant and the parental viruses were carried out in Vero cells in- oculated with an equal m.o.i. of each virus (Fig. 1). Although slower growth of the chimeric virus was observed at 24 h post-infection, it grew to a titre similar to the parental viruses by 36 h post-infection and remained the same on further incubation. Both the kinetics and Fig. 1. Growth of different viruses in tissue culture. Growth rates of viruses were determined under multi-step growth conditions (m.o.i. of ~0.01) for normal RPV, PPRV and the chimeric RPV– PPRN virus in Vero cells. Results are shown as the mean of duplicate readings. 2021