Background

In virology, the reverse genetics system is a technology for generating recombinant viruses that enables the manipulation of viral genes. Using this system, we can define the function of viral genes/proteins and investigate the mechanisms of viral pathogenicity [1]. To date, infectious viral cDNA clones from bacterial artificial chromosomes and plasmids or from in vitro ligation of viral cDNA fragments have been used for reverse genetics in studies on positive-sense single-stranded RNA (ssRNA) viruses [2–5]. Although these methods allow for the generation of recombinant viruses, they are time-consuming and involve complicated procedures. Thus, a simple and efficient reverse genetics system is required, particularly in the context of pandemics caused by emerging/re-emerging ssRNA viruses.

A method for the generation of recombinant ssRNA viruses using circular polymerase extension reaction (CPER) has been developed [6,7]. In this method, full-length viral cDNA fragments with overlapping ends and an RNA Pol-II promoter-encoding linker are assembled into a circular genome by DNA polymerase and are directly transfected into cells to recover infectious viruses. This technology was applied to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which possesses one of the largest genomes (~30 kb) among the ssRNA viruses, enabling the rapid generation of recombinant viruses. It also enabled the rapid characterization of SARS-CoV-2 variants of concern and variants of interest after their emergence [8–12].

Flaviviruses and SARS-CoV-2 perform translation in a cap-dependent manner, whereas certain ssRNA viruses, such as hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV), perform cap-independent internal ribosome entry site (IRES)-mediated translation. The CPER method, optimized for efficient capping after viral RNA transcription, may not be directly applicable to ssRNA viruses that use IRES-mediated translation. Therefore, we adapted the CPER method using a gene cassette containing an RNA Pol-I promoter and terminator sequence that is needed for the efficient translation of RNA viruses [13].

BVDV, now renamed as Pestivirus bovis, belongs to the Pestivirus genus of the Flaviviridae family and possesses ssRNA that undergoes IRES-mediated translation. BVDV is the causative agent of bovine viral diarrhea, which leads to economic losses in agriculture. Generating recombinant BVDV using conventional reverse genetics has proven challenging for three reasons: i) the viral sequence exhibits toxicity in bacteria [14]; ii) gene duplication in some BVDV strains makes it difficult to obtain the correct cDNA clones [15]; and iii) MDBK cells, the most common cell line for BVDV propagation, show high susceptibility but are difficult to transfect with nucleotides [16].

In this protocol, we describe an optimized CPER method for ssRNA viruses using IRES-mediated translation, with BVDV serving as an example. Our method overcomes commonly encountered challenges and successfully produces recombinant viruses over a short timeframe. In addition, we demonstrate the generation of recombinant viruses without the need for isolation from permissive cell cultures, which is useful for the rapid characterization of viral strains, particularly during a pandemic.