Plasmids pPol I-LZH, pPol I-MZH, and pPol I-SZH carrying the L, M and S segments of ZH548, respectively, were cloned in the plasmid pRF108, [24, 36] . The plasmid pPol I-SZHDNSs, derived from pPol I-SZH, carries two BbsI cloning sites in place of NSs [24] . The humanized Renilla reniformis luciferase sequence from phRL-SV40 (Promega, Charbonnières-les-Bains, France) was inserted in pPol I-SZHDNSs to give pPol I-SZHDNSs-hRLuc. The structure of pPol I-SZHDNSs-hRLuc plasmid was confirmed by sequence analysis. Recombinant rZHDNSs-GFP [24] and rZHDNSs-hRLuc RVFV stocks were produced under BSL3 conditions. Approximately 5610 5 BHK21/T7 cells were seeded in triplicate in 35 mm culture dishes. The following day, they were combined with FuGENEH6 transfection reagent (Roche Applied Science, Indianapolis, IN) and 0.5 mg each of pTM1-L and pTM1-N [25] , and 1 mg each of pPol I-LZH, pPol I-MZH, and either pPol I-SZHDNSs-GFP or pPol I-SZHDNSs-hRLuc in OptiMEM (Gibco, invitrogen, Carslbad, CA). One day later, the medium was renewed. Five days later, the supernatant containing the rescued virus was collected and stored at 280uC. To produce