Viable porcine arteriviruses with deletions proximal to the 3 ' end of thegenome

In order to obtain attenuated live vaccine candidates of porcine reproducti ve and respiratory syndrome virus (PRRSV), a series of deletions was introd uced at the 3' end of the viral genome using an infectious cDNA clone of th e Lelystad virus isolate. RNA transcripts from the full-length cDNA clones were transfected into BHK-21 cells. The culture supernatant of these cells was subsequently used to infect porcine alveolar macrophages to detect the production of progeny virus. It is shown that C-terminal truncation of the nucleocapsid (N) protein, encoded by ORF7, was tolerated for up to six amin o acids without blocking the production of infectious virus. Mutants contai ning larger deletions produced neither virus nor virus-like particles conta ining viral RNA. Deletion analysis of the 3' UTR immediately downstream of ORF7 showed that infectious virus was still produced after removal of seven nucleotides behind the stop codon of ORF7. Deletion of 32 nucleotides in t his region abolished RNA replication and, consequently, no infectious virus was formed. Serial passage on porcine alveolar macrophages demonstrated th at the viable deletion mutants were genetically stable at the site of mutat ion. In addition, the deletions did not affect the growth properties of the recombinant viruses in vitro, while their antigenic profiles were similar to that of wild-type virus. Immunoprecipitation experiments with the six-re sidue N protein-deletion mutant confirmed that the truncated protein was in deed smaller than the wild-type IN protein. The deletion mutants produced i n this study are interesting candidate vaccines to prevent PRRS disease in pigs.