Unraveling the complexity of PRRSV-2 recombination: recombination frequency, replication efficiency, and backbone fitness define highly recombinogenic backbone and evolutionary bottlenecks.

Recombination is a primary driver of porcine reproductive and respiratory syndrome virus (PRRSV) evolution. Currently, lineage 1 NADC30-like virus (L1C) and lineage 8 HP-PRRSV (L8E) recombinants dominate in China, whereas recombinants harboring the lineage five backbone are infrequently detected. To elucidate the mechanisms underlying this disparity, we investigated the interplay between recombination frequency, viral fitness, and strain-specific phenotypes using co-infection models. Next-generation sequencing (NGS) revealed that the lineage 5 replicase is prone to template switching, generating high frequencies of recombination junctions comparable to NADC30-like strains when viral RNA abundance is high. However, plaque purification demonstrated a disconnection between the generation of recombination events and the survival of viable progeny. While NADC30-like strains acted as a highly recombinogenic backbone, yielding up to 100% recombinant progeny, lineage 5-based recombinants were largely eliminated due to competitive exclusion. Furthermore, although a mutation known to reduce recombination frequency (K541R) successfully reduced errors at the nucleic acid level, it failed to prevent the emergence of recombinants under strong selective pressure. Our findings suggest that PRRSV recombination is not merely a stochastic event but a complex outcome dictated by strain-specific phenotypic profiles. The scarcity of lineage 5 recombinants is driven by a phenotypic deficit, which is a combination of replication efficiency and susceptibility to host clearance. This study highlights the dominant role of NADC30-like strains in driving viral diversity and underscores that vaccine safety depends on the holistic phenotypic fitness of the virus.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) recombination poses a significant threat to the farm as it accelerates viral evolution and leads to changes in pathogenicity and immune protection. Understanding why certain strains recombine frequently while others do not is critical for reducing this risk. This study unravels the complexity of PRRSV recombination, showing that it is determined by the specific phenotypic profile of the virus strains involved. We discovered that current epidemic strains (NADC30-like) act as highly recombinogenic backbones due to their biological traits. Surprisingly, classical vaccine strains (lineage 5) infrequently form viable recombinants not because they are genetically accurate, but because their distinct phenotypic weaknesses lead to their elimination by the host or competitors. This finding shifts the paradigm of vaccine safety, emphasizing that preventing recombination requires a holistic approach that considers the complex interplay of viral fitness and host adaptation, thereby providing critical guidance for the industry.