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

• Published in: Journal of general virology 2001-11, Vol.82 (11), p.2607-2614
• Main Authors: Verheije, M.H, Kroese, M.V, Rottier, P.J.M, Meulenberg, J.J.M
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions; amino acids; Animals; Base Sequence; Cells, Cultured; clones; Cloning, Molecular; complementary DNA; DNA, Complementary - genetics; genome; Genome, Viral; ID Lelystad, Institute for Animal Science and Health; ID-Lelystad, Instituut voor Dierhouderij en Diergezondheid; live vaccines; macrophages; Macrophages, Alveolar - virology; Molecular Sequence Data; mutants; mutation; nucleocapsid; Nucleocapsid Proteins - chemistry; Nucleocapsid Proteins - genetics; nucleotides; Open Reading Frames; porcine reproductive and respiratory syndrome; Porcine Reproductive and Respiratory Syndrome - virology; Porcine reproductive and respiratory syndrome virus; Porcine respiratory and reproductive syndrome virus; Porcine respiratory and reproductive syndrome virus - genetics; Porcine respiratory and reproductive syndrome virus - growth & development; Porcine respiratory and reproductive syndrome virus - pathogenicity; precipitin tests; progeny; RNA replication; Sequence Analysis, DNA; Sequence Deletion; stop codon; Swine; Transfection; virus-like particles; viruses
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/0022-1317-82-11-2607

In order to obtain attenuated live vaccine candidates of porcine reproductive and respiratory syndrome virus (PRRSV), a series of deletions was introduced at the 3' end of the viral genome using an infectious cDNA clone of the 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 amino acids without blocking the production of infectious virus. Mutants containing larger deletions produced neither virus nor virus-like particles containing 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 this region abolished RNA replication and, consequently, no infectious virus was formed. Serial passage on porcine alveolar macrophages demonstrated that the viable deletion mutants were genetically stable at the site of mutation. 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-residue N protein-deletion mutant confirmed that the truncated protein was indeed smaller than the wild-type N protein. The deletion mutants produced in this study are interesting candidate vaccines to prevent PRRS disease in pigs.