Application of the CPER reverse genetics system for genetic engineering of rabies virus

Reverse genetics (RG) systems are essential tools for basic virological studies and applied studies using engineered recombinant viruses in various research fields. While the circular polymerase extension reaction (CPER) has been widely applied to prepare a full-length infectious complementary DNA (cDNA) of positive-sense RNA viruses, its use for negative-sense RNA viruses (mononegaviruses) remains limited. Here, we report the first CPER-based RG system for rabies virus (RABV), a member of mononegaviruses. Infectious RABV was successfully rescued from cells transfected with helper plasmids and the CPER product, the assembled overlapping DNA fragments encoding the full-length viral genome cDNA and regulatory elements. Using this system, we generated wild-type, point-mutant, reporter-expressing, and chimeric RABVs, all of which retained their expected biological properties. Deep sequencing revealed that CPER-derived viruses occasionally harbor low-frequency mutations undetectable by Sanger sequencing, highlighting PCR-related artifacts as a limitation. In addition, CPER products with a pUC19 backbone could be directly applied for <i>Escherichia coli</i> transformation and cloning of RABV full-genome cDNA plasmids, offering a flexible, ligase-free cloning strategy for conventional RG. Our work establishes CPER as a versatile platform for engineering recombinant RABVs, facilitating rapid generation and genetic manipulation of RABV with potential applications for research on other mononegaviruses.IMPORTANCEReverse genetics systems allow researchers to generate recombinant viruses with precise genetic modifications, advancing studies of viral replication, pathogenicity, and vaccine development. However, constructing a full-length viral genome expressing plasmids is often time-consuming and technically demanding. To bypass the cloning process, a simple, cloning-free reverse genetics platform based on the circular polymerase extension reaction (CPER) has been applied for positive-sense RNA viruses. In this study, we applied the CPER-based reverse genetics system for rabies virus (RABV), a mononegavirus, enabling rapid and flexible generation of recombinant RABVs, including mutant, reporter-expressing, and chimeric clones. Our approach greatly facilitates genetic engineering of RABV and provides a versatile framework that can be extended to other mononegaviruses, thereby accelerating both basic and applied virology research.