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Title: Development of a genome copy specific RT qPCR assay for divergent strains of type II porcine reproductive and respiratory syndrome virus

Author
item Spear, Allyn
item Faaberg, Kay

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2015
Publication Date: 6/15/2015
Publication URL: http://handle.nal.usda.gov/10113/61088
Citation: Spear, A., Faaberg, K.S. 2015. Development of a genome copy specific RT qPCR assay for divergent strains of type II porcine reproductive and respiratory syndrome virus. Journal of Virological Methods. 218:1-6.

Interpretive Summary: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of disease and production loss in both the US and global pork industry. This virus changes very rapidly and has caused many new outbreaks in the past 25 years. Traditional methods for making vaccines have not worked well in protecting livestock, so more basic research into how different strains of the virus cause disease is needed. One challenge to this research is the difficulty in accurately comparing levels of virus replication between different strains and in different organs of the same animal. We have developed a method that will allow researchers to very accurately determine how much virus is being made in any organ and correctly compare these numbers between many different strains of PRRSV. This method will also allow any PRRSV researcher to easily adapt this strategy to their specific strains of virus, or to adapt to new strains of PRRSV that may appear in future outbreaks.

Technical Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) became a significant pathogen of swine upon its emergence in the late 1980’s and since then has exemplified a rapidly evolving, constantly reemerging 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 our 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. We have developed an RT qPCR based technique 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 II PRRSV strains in GenBank for which nsp9 sequence is available. We demonstrate that the assay is linear over eight orders of magnitude (10^10 to 10^2 copies) and can be readily adapted for multiplex detection of additional divergent PRRSV strains. This assay will add significantly to our 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.