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We generated novel recombinant influenza A viruses (vNA38) harboring dicistronic NA segments with an extended native 5′ terminal sequence of 70 nucleotides comprised of the last 42 nucleotides of the NA ORF and the 5′ noncoding region (5′ NCR). vNA38 viruses replicated stably and more efficiently than vNA35 viruses with a dicistronic NA segment comprised of the native 5′ NCR only, that we described previously (Vieira Machado, A., Naffakh, N., van der Werf, S., Escriou, N., 2003. Expression of a foreign gene by stable recombinant influenza viruses harboring a dicistronic genomic segment with an internal promoter. Virology 313, 235–249). In addition, vNA38 viruses drove the expression of higher levels of encoded heterologous proteins than corresponding vNA35 viruses, both in cell culture and in the pulmonary tissue of infected mice. These data demonstrate that a sequence overlapping 5′ coding and noncoding regions of the NA segment determines efficient replication and/or propagation of the vRNA. Intranasal immunization of mice with live vNA38 viruses induced B and T cell responses specific for the heterologous protein expressed, establishing the usefulness of such recombinant influenza viruses with a dicistronic segment for the development of live bivalent vaccines.

Based on the observation that an internally located 3′ promoter sequence can be functional (R. Flick and G. Hobom, Virology, 1999, 262(1), 93–103), we generated transfectant influenza A viruses harboring a dicistronic segment containing the CAT gene (660 nt) or a fragment of the Mengo virus VP0 capsid gene (306 nt) under the control of a duplicated 3′ promoter sequence. Despite slightly reduced NA expression, the transfectant viruses replicated efficiently and proved to be stable upon both serial passage in vitro in MDCK cells and in vivo replication in the pulmonary tissue of infected mice. Internal initiation of replication and transcription from the second, internal, 3′ promoter directed the synthesis of subgenomic vRNA and mRNA and therefore permitted expression of the foreign gene product, e.g., the CAT enzyme. The design of this vector may prove particularly appropriate for the utilization of influenza virus for the expression of heterologous proteins in their native form.