A PCR-based protocol for generating West Nile virus replicons

A new protocol for the generation of West Nile virus (WNV) replicons was developed. Fragmented cDNAs that covered the entire WNV RNA sequence, except the sequence corresponding to nucleotides 190–2379, were amplified separately by polymerase chain reactions (PCRs) using primer set franking with overlapping sequences of 40–50 bp at the 5′- and the 3′-ends of each fragment. All amplified fragments were mixed together and annealed to each other at the overlapping sequences. The annealed-DNA fragments were elongated by DNA polymerase and amplified by short-cycle PCRs to generate full-sized WNV replicon cDNAs. The WNV replicons were transcribed in vitro using the replicon cDNAs as templates. When the in vitro-transcribed replicon was introduced into mammalian cells, the viral envelope protein and viral positive- and negative-strand RNAs were detected in the replicon-transfected cells. It is noteworthy that the synthesis of the replicon cDNAs and the replicons took just 1 week, and that the use of a high-fidelity DNA polymerase afforded stability to the sequence of the synthetic replicon.