Establishment of a circular polymerase extension reaction–based reverse genetics system for porcine reproductive and respiratory syndrome virus

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Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses in the global swine industry. Reverse genetics is an essential approach for elucidating viral replication mechanisms, pathogenicity, and vaccine design; however, existing reverse genetics systems for PRRSV are often technically demanding and time-consuming due to the large and genetically unstable viral genome. Here, we established a reverse genetics system for PRRSV based on the circular polymerase extension reaction (CPER), a bacteria-free method for assembling full-length viral genomes. Infectious PRRSV was successfully rescued following transfection of CPER-assembled DNA into human embryonic kidney-derived HEK293T cells. The rescued virus exhibited growth kinetics and plaque morphology comparable to those of the parental strain in porcine alveolar macrophage-derived PAM-T43 cells. To demonstrate the applicability of this system for genetic manipulation, targeted synonymous mutations were introduced into the ORF5 gene, and the resulting mutant virus was successfully recovered and genetically confirmed. These results indicate that the CPER-based reverse genetics system provides a rapid and flexible platform for generating genetically defined PRRSV variants. This approach is expected to facilitate studies on PRRSV pathogenesis, antigenic variation, and vaccine development, thereby contributing to improved control strategies for this economically important swine disease.

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