Fig. 1.

Summary illustrating the recent innovations in experimental design of rescuing recombinant non-segmented negative-sense RNA viruses. A, B Design of the full-length cDNA antigenome flanked by trans and cis-acting ribozymes to generate the correct 5′ and 3′-ends in T7 and CMV promoter-driven systems for initial RNA transcription. The strategy in A requires exogenous T7 RNA polymerase while B is free of exogenous T7 RNA polymerase and relies on the CMV promoter. C The two-plasmid system design with a single helper plasmid encoding three translational cassettes of the essential viral replication proteins (N, P, and L). Each translational cassette is spanned with an appropriate promoter (T7 or CMV) and terminator (T7T or poly-A tail) that are dependent on the rescue strategy and with or without exogenous T7 RNA polymerase. In this system, only the plasmid containing the full-length antigenome and the single helper plasmids will be co-transfected to generate an infectious virus. D The design of the one plasmid and helper plasmid free based-system that implements both T7 and CMV promoter-driven systems with or without exogenous T7 RNA polymerase. In this system, additional promoter (T7 or CMV) sequences are inserted by careful substitution into the viral cDNA at strategic positions. This allows transcription of sub-genomic RNAs that encode essential viral replication proteins (N, P, and L) that are needed for the RNP complex to form. The T7 promoter-based system requires an exogenous T7 RNA polymerase and the CMV promoter-based system is free of exogenous T7 RNA polymerase.