Expression of a foreign epitope by porcine reproductive and respiratory syndrome virus

The potential of porcine reproductive and respiratory syndrome virus (PRRSV ) as a viral vector was explored by the insertion of a sequence encoding a foreign antigen into the infectious cDNA clone of the Lelystad virus isolat e. An epitope of the hemagglutinin (HA) protein of human influenza A virus was introduced at the 5' end and at the 3' end of ORF7, in each case result ing in a fusion protein between the HA epitope and the nucleocapsid (N) pro tein. Furthermore, in the construct carrying the HA sequences at the 5' end of ORF7, additional in-frame insertions encoding the autoprotease 2A of fo ot-and-mouth disease virus were made between the HA and ORF7 sequences to e nsure the generation of a functional N protein from its hybrid precursor. W hen RNA transcripts from these full-length cDNA clones were transfected int o BHK-21 cells, they were each found to replicate, to express the HA epitop e, and to produce progeny virus. However, fusion of the HA epitope directly to the nucleocapsid protein either at the N terminus or at the C terminus adversely affected both the viability and the genetic stability of the reco mbinant PRRS viruses. Serial passage of the recombinant viruses on porcine alveolar macrophages demonstrated that these viruses had lost (part of) the HA epitope at passage four. In contrast, in the PRRS viruses expressing th e HA epitope from a precursor cleavable by the autoprotease 2A peptide, the HA epitope was still intact after four passages, and no effect on the viab ility of these viruses was observed. Immunoprecipitation and pulse chase ex periments revealed the efficient and presumably cotranslational cleavage of the HA epitope from the N protein by the 2A protease. Our results demonstr ate the feasibility of using PRRSV as a viral vector that might be suitable for the delivery of antigens from other pathogens to the immune system of the pig. (C) 2000 Academic Press.