Reverse genetics for peste-des-petits-ruminants virus (PPRV): promoter and protein specificities.

Peste-des-petits-ruminants virus (PPRV) (family Paramyxoviridae, genus Morbillivirus) causes an acute febrile illness in sheep and goats resulting in significant morbidity and mortality in infected herds. The paramyxoviruses all have negative sense, non-segmented RNA genomes and their host range and pathogenic determinants have been extensively studied using reverse genetics. This technology also enables a more rational approach to be taken with respect to vaccine design. In order to initiate this type of work for PPRV we constructed a PPRV minigenome and studied its expression in transfected cells. As for other morbilliviruses, the minimum requirements for minigenome rescue were shown to be the cis-acting elements of the genome (GP) and antigenome (AGP) promoters as well as the three trans-acting helper proteins N (nucleocapsid), P (phosphoprotein) and L (large polymerase). Homologous PPRV helper proteins were compared to their heterologous analogues from the closely related rinderpest virus (RPV) and heterologous minigenome rescue was found to be a much less efficient process. By engineering two GP/AGP chimeric minigenomes we also identified differences between the two viruses in the specific interactions between the promoters and the transcriptase/replicase complexes. The PPRV minigenome was also shown not to strictly comply with the "rule of six"in vitro.