Virus Research 163 (2012) 98–107 Contents lists available at SciVerse ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Genetic and phenotypic characterization of a 2006 United States porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field  Phillip C. Gauger a , Kay S. Faaberg b , Baoqing Guo a , Matthew A. Kappes b , Tanja Opriessnig a,∗ a Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA b Virus and Prion Research Unit, National Animal Disease Center-USDA-Agricultural Research Service, Ames, IA, USA article info Article history: Received 8 February 2011 Received in revised form 26 August 2011 Accepted 30 August 2011 Available online 7 September 2011 Keywords: Porcine reproductive and respiratory syndrome virus (PRRSV) In vitro characterization Sequencing 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 demon- strated slower replication in vitro compared to the three other isolates and grew to a peak titer of 5.4 × 10 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 diame- ter 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 variable levels of 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 pat- tern. 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 demon- strated marked nucleotide variability within non-structural protein (nsp) 1 and nsp2, and a nucleotide deletion of 24 bases in nsp2. Sequence homology with VR-2332 and MN184 was 88.4% and 82.9%, respec- tively; 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 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. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive sense, enveloped, single-stranded RNA virus of the fam- ily Arteriviridae in the order Nidovirales (Cavanagh, 1997). PRRSV is the cause of porcine reproductive and respiratory syndrome (PRRS), first reported in the United States in 1987 (Keffaber, 1989) and in Europe in 1990 (Paton et al., 1991). The virus was isolated from swine shortly thereafter and is currently ubiquitous in most swine populations (Cho and Dee, 2006; Collins et al., 1992; Shi et al., 2010; Wensvoort et al., 1991). Clinical manifestations of PRRS include sys- temic and respiratory disease in growing pigs and reproductive  Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. ∗ Corresponding author. Tel.: +1 515 294 1137; fax: +1 515 294 3564. E-mail address: tanjaopr@iastate.edu (T. Opriessnig). failure in naïve breeding animals (Halbur et al., 1996; Mengeling et al., 1998). PRRS is the most economically significant porcine dis- ease worldwide due to increased mortality and reduced growth performance (Neumann et al., 2005). Genetic analysis has established two predominant PRRSV geno- types; the European genotype (strain Lelystad; Type 1) and the North American genotype (strain VR-2332; Type 2) which share approximately 60% nucleotide identity (Allende et al., 1999; Nelsen et al., 1999). Historically, swine herds in the United States have been predominately infected with Type 2 viruses; however, since 1999 Type 1 isolates have been identified as well (Dewey et al., 2000; Fang et al., 2004; Ropp et al., 2004). Genetic divergence is also evi- dent within each genotype where individual PRRSV isolates can vary up to approximately 20% on the nucleotide level (Han et al., 2006). Due to extensive genetic and antigenic diversity and the presence of multiple genotypes circulating within the swine popu- lation, PRRSV is difficult to control through the use of vaccines due to the lack of cross protection among genetically dissimilar viruses (Kimman et al., 2009). 0168-1702/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2011.08.017