Journal of General Virology (2007), 88, 2019–2027 DOI 10.1099/vir.0.82913-0 Correspondence Satya Parida satya.parida@bbsrc.ac.uk Received 9 February 2007 Accepted 19 March 2007 Rescue of a chimeric rinderpest virus with the nucleocapsid protein derived from peste-des-petits-ruminants virus: use as a marker vaccine Satya Parida, Madhuchhanda Mahapatra, Sai Kumar, Subash C. Das, Michael D. Baron, John Anderson and Thomas Barrett Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 0NF, UK The nucleocapsid (N) protein of all morbilliviruses has a highly conserved central region that is thought to interact with and encapsidate the viral RNA. The C-terminal third of the N protein is highly variable among morbilliviruses and is thought to be located on the outer surface and to be available to interact with other viral proteins such as the phosphoprotein, the polymerase protein and the matrix protein. Using reverse genetics, a chimeric rinderpest virus (RPV)/ peste-des-petits-ruminants virus (PPRV) was rescued in which the RPV N gene open reading frame had been replaced with that of PPRV (RPV–PPRN). The chimeric virus maintained efficient replication in cell culture. Cattle vaccinated with this chimeric vaccine showed no adverse reaction and were protected from subsequent challenge with wild-type RPV, indicating it to be a safe and efficacious vaccine. The carboxyl-terminal variable region of the rinderpest N protein was cloned and expressed in Escherichia coli. The expressed protein was used to develop an indirect ELISA that could clearly differentiate between RPV- and PPRV-infected animals. The possibility of using this virus as a marker vaccine in association with a new diagnostic ELISA in the rinderpest eradication programme is discussed. INTRODUCTION Rinderpest, or cattle plague, is an economically important disease of domestic and wild ruminants. The disease is caused by rinderpest virus (RPV), which is classified in the genus Morbillivirus in the family Paramyxoviridae. The morbillivirus genome consists of a single strand of negative- sense RNA, which is organized into six contiguous, non- overlapping transcription units encoding six structural proteins, the nucleocapsid (N), polymerase (P), matrix (M), fusion (F), haemagglutinin (H) and large (L) proteins, in the order 39-N-P-M-F-H-L-59 (Bailey et al., 2005; Crowley et al., 1988; Rima et al., 1986). In addition, there are two non-structural proteins (C and V), which are translated from the P gene open reading frame (ORF) by co-transcriptional RNA editing (V) or from an overlapping ORF (C) (Bellini et al., 1985; Cattaneo et al., 1989; Mahapatra et al., 2003). The RNA genome is encapsidated and protected by N protein and, like many viruses in the family Paramyxoviridae, it complies with the ‘rule of six’, i.e. the genomes must be an exact multiple of six, as each N protein molecule is thought to interact with six nucleotides (Calain & Roux, 1993). Computer alignment of morbilli- virus N protein sequences has defined regions with varying degrees of homology: the N-terminal part of the protein is 0008-2913 G 2007 SGM Printed in Great Britain very well conserved, whilst the C-terminal region is found to vary greatly among related viruses (Diallo et al., 1994). The N protein is the most abundant of the structural proteins by virtue of its position close to the promoter (Cattaneo et al., 1987a, b) and it performs several functions during virus replication. These include encapsidation of the genomic RNA into an RNase-resistant ribonucleocapsid (the template for RNA synthesis) and association with the P/L polymerase complex (required for transcription and replication); it also appears to interact with the M protein during virus assembly (Buchholz et al., 1994; Coronel et al., 2001; Curran, 1996; Curran et al., 1998; Ryan & Portner, 1990). We were interested in finding out whether this protein, with such diverse interactions with other viral proteins, could be substituted between closely related morbilliviruses of ruminant species, namely RPV and peste-des-petits-ruminants virus (PPRV). In this study, we report the generation and characterization of a chimeric RPV where the N protein gene of the RPV vaccine strain was replaced with the equivalent gene from the PPRV vaccine. The recombinant virus exhibited growth char- acteristics in cell culture similar to those of the parental viruses, and animals vaccinated with this chimeric virus were protected from challenge with virulent virus. 2019