Molecular Evolution of Porcine Reproductive and Respiratory Syndrome Virus Field Strains from Two Swine Production Systems in the Midwestern United States from 2001 to 2020.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses an extensive economic threat to the United States swine industry. The high degree of PRRSV genetic and antigenic variability challenges existing vaccination programs. We evaluated the ORF5 sequence of 1,931 PRRSV-2 strains detected from >300 farms managed by two pork production systems in the midwestern United States from 2001 to 2020 to assess the genetic diversity and molecular characteristics of heterologous PRRSV-2 strains. Phylogenetic analysis was performed on ORF5 sequences and classified using the global PRRSV classification system. N-glycosylation and the global and local selection pressure in the putative GP5 encoded by ORF5 were estimated. The PRRSV-2 sequences were classified into lineage 5 (L5;  = 438[22.7%]) or lineage 1 (L1;  = 1,493[77.3%]). The L1 strains belonged to one of three subclades: L1A ( = 1,225[63.4%]), L1B ( = 69[3.6%]), and L1C/D ( = 199[10.3%]). 10 N-glycosylation sites were predicted, and positions N44 and N51 were detected in most GP5 sequences ( = 1,801[93.3%]). Clade-specific N-glycosylation sites were observed: 57th in L1A, 33rd in L1B, 30th and 34th in L1C/D, and 30th and 33rd in L5. We identified nine and 19 sites in GP5 under significant positive selection in L5 and L1, respectively. The 13th, 151st, and 200th positive selection sites were exclusive to L5. Heterogeneity of N-glycosylation and positive selection sites may contribute to varying the evolutionary processes of PRRSV-2 strains circulating in these swine production systems. L1A and L5 strains denoted excellence in adaptation to the current swine population by their extensive positive selection sites with higher site-specific selection pressure. Porcine reproductive and respiratory syndrome virus (PRRSV) is known for its high genetic and antigenic variability. In this study, we evaluated the ORF5 sequences of PRRSV-2 strains circulating in two swine production systems in the midwestern United States from 2001 to 2020. All the field strains were classified into four major groups based on genetic relatedness, where one group is closely related to the Ingelvac PRRS MLV strain. Here, we systematically compared differences in the ORF5 polymorphisms, N-glycosylation sites, and local and global evolutionary dynamics between different groups. Sites 44 and 51 were common for N-glycosylation in most amino acid sequences ( = 1,801, 93.3%). We identified that the L5 sequences had more positive selection pressure compared to the L1 strains. Our findings will provide valuable insights into the evolutionary mechanisms of PRRSV-2 and these molecular changes may lead to suboptimal effectiveness of Ingelvac PRRS MLV vaccine.