Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPV KO ) can be rescued. A recombinant virus, rRPV KO L-RREGFPR, which grew at an indistinguishable rate and to an identical titer as rRPV KO in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 10 4 TCID 50 of the rRPV KO parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPV KO L-RREGFPR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo.Morbilliviruses cause significant levels of disease in humans and animals throughout the world. For example, the human pathogen measles virus (MV) infects over 40 million individuals per annum and leads to the death of around 800,000 children. The classical course of disease progression is difficult to mimic comprehensively in tractable animal models as the virus has a highly restricted host range. A number of nonhuman primate models have been developed which reproduce many, but not all, aspects of the human disease. However, each has some limitations and they are all dead-end hosts (2,45,47). This limitation has led to the development of a range of smallanimal models, which are useful but tend to reflect only limited aspects of the human disease (29,30,33,37,40). These models are augmented by others which use closely related morbilliviruses, such as rinderpest virus (RPV) or canine distemper virus (CDV), to infect natural hosts such as cattle and ferrets (18, 49, 52). Although many aspects of MV infection of humans are mirrored in the pathogenesis of CDV and RPV, each has its characteristic pattern of disease progression. Thus, wild-type CDV is much more likely to infect the central nervous system than MV, whereas RPV has not been found to infect the central nervous system. In contrast, wild-type RPV has a particular propensity to infec...