title: Development of a genome copy specific RT-qPCR assay for divergent strains of type 2 porcine reproductive and respiratory syndrome virus.
abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) became a significant pathogen of swine upon its emergence in the late 1980s and since then has exemplified a rapidly evolving, constantly re-emerging pathogen. In addition to the challenges faced in development of vaccines and diagnostics, research on the basic molecular pathogenesis of PRRSV is also restrained by the ability to accurately and comparatively quantitate levels of replication in different tissues and between strains. This is further complicated by the presence of non-genomic RNA within infected tissues which are generally detected with equivalent efficiency by RT-qPCR based techniques, thereby introducing inherent error in these measurements that may differ significantly by tissue and strain. To address this, an RT-qPCR based technique was developed which targets the viral RNA-dependent RNA polymerase gene (nsp9) which is unique to genomic RNA, being absent from all subgenomic and heteroclite RNAs. This assay targets a region of considerable sequence conservation, and based on sequence only, should be quantitative for approximately 40% of all Type 2 PRRSV strains in GenBank for which nsp9 sequence is available. The assay was demonstrated to be linear over nine orders of magnitude (10(10)-10(2) copies) and can be readily adapted for multiplex detection of additional divergent PRRSV strains. This assay will add significantly to the ability to assess and compare PRRSV replication in a variety of tissues and between divergent strains, including highly pathogenic strains of considerable concern to the global pork industry.
authors.affiliation: School of Freshwater Sciences, United States Department of Agriculture, Agricultural Research Service, 600 E. Greenfield Avenue, Milwaukee, WI 53204, USA. Electronic address: allyn.spear@ars.usda.gov.
authors.forename: Allyn
authors.initials: A
authors.lastname: Spear
authors.affiliation: National Animal Disease Center, United States Department of Agriculture, Agricultural Research Service, 1920 Dayton Avenue, Ames, IA 50010, USA.
authors.forename: Kay S
authors.initials: KS
authors.lastname: Faaberg
mesh_terms: D000818:Animals; D016679:Genome, Viral / Q000235:genetics; D057230:Limit of Detection; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000145:classification* / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase
keywords: Genome copy; PRRSV; RT-qPCR; nsp9