title: Insertion and deletion in a non-essential region of the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome (PRRS) virus: effects on virulence and immunogenicity.
abstract: Developing a vaccine that can differentiate infected and vaccinated animals (DIVA) is a new challenge in the design of a vaccine for porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein 2 (nsp2) is the single largest viral product, and it has multiple roles in polypeptide processing and replication complex formation. Using reverse genetics and an infectious PRRSV cDNA clone, we constructed several deletion mutants in the non-essential region of nsp2. One mutant, which has a 131 amino acid deletion within a relatively conserved region of nsp2, was recovered and found to produce a viable virus. The deleted region was replaced with a peptide tag encoding eight amino acids. A recombinant virus containing the 131 amino acid deletion was found to produce normal virus yields in MARC-145 cells and porcine alveolar macrophages (PAM); however, gross and micro-histopathology showed that the virus was less virulent in pigs. The 131 amino acid peptide was expressed as a recombinant protein and used to coat enzyme-linked immunosorbent assay (ELISA) plates. This peptide was recognized by sera from pigs infected with wild-type virus, but not by sera from pigs infected with the deletion mutant. The results from this study show that nsp2 is an important target for the development of marker vaccines and for virus attenuation.
authors.affiliation: Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS, 66506, USA. dykim@utmb.edu
authors.forename: Dal-Young
authors.initials: DY
authors.lastname: Kim
authors.forename: Troy J
authors.initials: TJ
authors.lastname: Kaiser
authors.forename: Kyle
authors.initials: K
authors.lastname: Horlen
authors.forename: Marcia L
authors.initials: ML
authors.lastname: Keith
authors.forename: Lucas P
authors.initials: LP
authors.lastname: Taylor
authors.forename: Rika
authors.initials: R
authors.lastname: Jolie
authors.forename: Jay G
authors.initials: JG
authors.lastname: Calvert
authors.forename: Raymond R R
authors.initials: RR
authors.lastname: Rowland
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002460:Cell Line; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008168:Lung / Q000473:pathology; D016676:Macrophages, Alveolar / Q000821:virology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019318:Porcine Reproductive and Respiratory Syndrome / Q000473:pathology / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity*; D017384:Sequence Deletion; D013552:Swine; D022581:Vaccines, Marker / Q000276:immunology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology*; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000502:physiology*
chemical_list: D000914:Antibodies, Viral; D022581:Vaccines, Marker; D017361:Viral Nonstructural Proteins; D037521:Virulence Factors