Experimental evidence of vaccine-driven evolution of porcine reproductive and respiratory syndrome virus type 2

Despite extensive use of vaccination, porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) continues to evolve, likely driven by escape from natural or vaccine-derived immunity. However, direct evidence of vaccine-induced evolutionary pressure remains limited. Here, we tracked the evolution of PRRSV-2 sublineage 1A strain IA/2014 (variant 1A-unclassified) genome from infection chains of sequentially infected pigs under different immune conditions. Weaned pigs were divided into three groups: a non-immunized control group and two groups vaccinated with different modified live virus (MLV) vaccines, namely Prevacent® PRRS MLV (variant 1D.2) and Ingelvac PRRS® MLV (variant 5A.1). Sixty-four days post-vaccination, the pigs were challenged with IA/2014 PRRSV-2. Virus infection chains (which used serum from pigs in batch <i>n</i> to infect batch <i>n</i> + 1) were maintained across six sequential batches of roughly seven pigs each, allowing for virus evolution to occur across the ~ 84 days of the infection chain. A total of 110 serum samples were successfully sequenced. Vaccinated groups exhibited over twice the genetic divergence from the original challenge virus (0.3%-0.4% mean nucleotide distance) compared to non-immunized group (0.15%). Variability was concentrated in ORF1a and ORF1b. Deep sequencing revealed more rapid shifts of viral quasispecies composition in vaccinated pigs, and more homogeneous viral populations over batches compared to non-immunized pigs. Selection pressure analyses indicated strong purifying selection in one vaccinated group, though without clear signals at known antigenic sites in all treatment groups. However, vaccinated pigs had significantly higher cycle threshold values (<i>P</i><.001), indicating lower viral loads and suggesting potential fitness limitations for highly diverged viruses in immunized pigs. These findings demonstrate that MLV vaccination can exert substantial evolutionary pressure on PRRSV-2, driving genetic diversification and highlighting the need for continuous PRRS monitoring and adaptive control strategies.