Single-step construction of a picornavirus replicon RNA with precise ends

A versatile single-step method is described for constructing a picornavirus replicon RNA with precise ends to facilitate improved understanding of viral genome function and mimic native virus replication in host cells as far as possible. The key innovation in this new approach is the use of a bridge primer to both introduce a ribozyme sequence for cis-cleavage of RNA to generate precise 5′ ends of EV71 RNA and also mediate overlapping assembly of two fragments. Using an EV71 replicon as a test case, precise ends for the viral replicon were shown to be important for efficient virus replication. Thus, our work provides a novel efficient way to generating higher efficient viral replicon with precise ends and this novel method can be applied to other picornaviruses' research.