Submitted to: Virus Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/31/2011
Publication Date: 1/1/2012
Citation: Gauger, P.C., Faaberg, K.S., Guo, B., Kappes, M.A., Opriessnig, T. 2012. Genetic and phenotypic characterization of a 2006 United States porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field. Virus Research. 163(1):98-107. Interpretive Summary: Porcine reproductive and respiratory syndrome virus (PRRSV) is the foremost disease of swine in the United States. In 2006, a swine herd from North Carolina experienced high morbidity (50%) and mortality (20%) and a novel isolate of PRRSV was isolated. The objective of this research was to characterize the isolate, NC16845, by growth kinetics, plaque morphology and RNA display, in comparison with 3 other well-characterized type 2 strains. In addition, we derived to complete genomic sequence of the isolate by a new technology referred to as large-scale parallel pyrosequencing. The majority of the nucleotide differences from the prototype strain, VR-2332, were identified in open reading frame (ORF) 1, specifically within a region known as the non-structural protein 2 (nsp2). Analysis identified significant nucleotide degeneracy and a discontinuous nucleotide deletion of 24 bases also within nsp2. In addition, at least two cocirculating quasispecies were identified. The novel isolate was found to represent a new subtype of PRRSV type 2 strains. The information obtained advances our understanding of porcine reproductive and respiratory syndrome virus evolution which aids the development of rationally designed vaccines.
Technical Abstract: The objective of this study was to characterize a porcine reproductive and respiratory syndrome virus (PRRSV) isolated from United States pigs experiencing high morbidity (50%) and mortality (20%). The PRRSV isolate, designated NC16845b, was characterized through phenotypic analysis and genomic sequencing and compared to Type 2 PRRSV isolates VR-2332, MN184 and VR-2385. NC16845b demonstrated slower replication in vitro compared to the three other isolates and grew to a peak titer of 5.4x10**5 plaque forming units (PFU) per ml at 60 h post inoculation, which was 4- to 13-fold less than the peak titer of the other three viruses. NC16845b plaques were intermediate size averaging 3.3 mm in diameter that was larger than MN184 plaques and smaller than VR-2385 and VR-2332. Using Northern blot analysis, viral and subgenomic RNA were detected that demonstrated slightly less hybridization in some open reading frames (ORF) compared to the other viruses. NC16845b is 15,389 nucleotides in length and ORF 5 restriction fragment length polymorphism (RFLP) analysis demonstrated a 1-18-2 pattern. Among all available Type 2 complete genome sequences, NC16845b showed the highest nucleotide homology (91.2%) to atypical PRRSV strain JA142. Compared to prototype VR-2332, NC16845b demonstrated marked nucleotide variability within non-structural protein (nsp) 1beta and nsp2, and a nucleotide deletion of 24 bases in nsp2. Sequence homology with VR-2332 and MN184 was 88.4% and 82.9%, respectively; homology with the ORF2-7 of VR-2385 was 90.4%. Collectively, these data indicate that, compared to prototype Type 2 PRRSV isolates, NC16845b exhibited slower in vitro replication and growth properties, had regions of heterogeneity within ORF1a that corresponded to at least two individual virus quasispecies, and also contained a continuous 8 amino acid deletion in the nsp2 protein.