The application of a PRRSV reverse genetic system for the study of nonstructural protein (nsp) function
dc.contributor.author | Kim, Dal-Young | |
dc.date.accessioned | 2007-05-08T17:01:37Z | |
dc.date.available | 2007-05-08T17:01:37Z | |
dc.date.graduationmonth | May | en |
dc.date.issued | 2007-05-08T17:01:37Z | |
dc.date.published | 2007 | en |
dc.description.abstract | Infectious cDNA clones of PRRSV make it possible to construct marker viruses for the study of virus replication and pathogenesis. The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the single largest protein produced during virus replication. The cDNA of the pCMV-129 infectious PRRSV clone was modified by creating unique Mlu I and SgrA I restrictions sites at nucleotide (nt) positions 3,219 and 3,614, respectively: both located within the C-terminal region of nsp2. cDNAs coding for oligo- and polypeptide tags, including FLAG, enhanced green fluorescent protein (EGFP) and firefly luciferase were inserted into the newly created restriction sites. The results showed that only the EGFP-containing genomes were properly expressed and produced virus. EGFP fluorescence, but not EGFP immunoreactivity, was lost during passage of recombinant EGFP viruses in culture. Sequencing of a fluorescence-negative EGFP virus showed that the EGFP remained intact, except for the appearance of mutations that may affect chromophore formation. The results show that nsp2 can be a site for the expression of foreign proteins. Removal of the region between Mlu I and SgrA I sites resulted in a virus that contained a 131 amino acid deletion. The deleted region was replaced with EGFP or an eight amino acid influenza hemagglutanin (HA) tag. Recombinant viruses were used to infect pigs. Gross and micro-histopathology showed reduced pathogenesis when compared to the parent wild-type virus. The 131 amino acid peptide, when expressed as a recombinant protein and coated onto enzyme linked immunosorbent assay (ELISA) plates, was recognized by sera from pigs infected with wild-type virus, but not the deletion mutants. The results from this study show that nsp2 is a potential target for the development of marker vaccines that can differentiate infected from vaccinated animals (DIVA) and for virus attenuation. | en |
dc.description.advisor | Raymond R. R. Rowland | en |
dc.description.degree | Doctor of Philosophy | en |
dc.description.department | Department of Diagnostic Medicine/Pathobiology | en |
dc.description.level | Doctoral | en |
dc.identifier.uri | http://hdl.handle.net/2097/317 | |
dc.language.iso | en_US | en |
dc.publisher | Kansas State University | en |
dc.subject | PRRSV | en |
dc.subject.umi | Biology, Veterinary Science (0778) | en |
dc.title | The application of a PRRSV reverse genetic system for the study of nonstructural protein (nsp) function | en |
dc.type | Dissertation | en |