Figure 1

Schematic of the fusion-PCR strategy used to generate viral chimeras. (A). Strategy used to generate fpMODV-WNV(prM-E) in seven steps. The approximate location of primers and intermediate PCR products are shown on each viral genome (not scaled). Note that just viral sequences are depicted, the actual MODV template was pACNR-FLMODV while WNV template was viral cDNA (see materials and methods). All intermediate products and primers are further described in accompanying Table 1. Chimeric primers are represented by bicolor arrows. Steps 1–4: Products MW1, MW2, MW3 and M4 were generated by PCR with the indicated primers. These fragments were used as construction blocks in subsequent steps in fusion PCRs. Step 5: Products MW1 and MW2 were fused amplifying with primers M-F1 and MW-R2 to generate product MW5. Step 6: MW5 was fused with MW3 using primers M-F1 and M-R3 to give MW6. Step 7: In the final reaction, a full-length chimeric product was generated by fusing MW6 to M4 using primers T7-MOD-F and M-R10600. (B). Maps of final constructs highlighting the resulting amino acid chimeric sequences. Arrows indicate protease cleavage sites. Sequences from the heterologous viruses (WNV or CxFV) are underlined.