Alterations in caspase cleavage motifs of NP and M2 proteins attenuate virulence of a highly pathogenic avian influenza virus.

The NP and M2 proteins of influenza A viruses are cleaved by caspases. M2 cleavage occurs with both human and avian viruses, whereas NP cleavage is specific for human strains. In the present study we have modified fowl plaque virus (A/FPV/Rostock/34 (H7 N1)) by introducing the NP cleavage site and removing the M2 cleavage site and have analyzed the effects of these modifications on virus growth and pathogenicity. The viruses generated by reversed genetics were: mutant NPgd which had the NP cleavage site METD downward arrowG(17), recombinant wild type virus and mutant NPdel with the non-cleavable motifs METGG(17) and MET-G(17), respectively, and mutant M2nn in which the M2 cleavage site VDVD downward arrowDG(89) of wild type virus was replaced by the non-cleavable sequence VNVNDG(89). Mutant NPgd replicated in cell cultures and in chicken embryos equally well as wild type virus and mutant NPdel. This observation was in contrast to previous results obtained with the human WSN strain that required caspase cleavage of NP for optimal virus growth. Thus, it appears that acquisition of the NP cleavage site is an adaptive mutation promoting interspecies transmission from an avian to a mammalian host. Like mutant NPgd, mutant M2nn virus did not show replication defects in cell culture and in chicken embryos. However, there was a reduction in pathogenicity for chickens, that was moderate with NPgd and dramatic with M2nn. Single injections with NPgd and M2nn at low doses induced high antibody titers and protected chickens from lethal FPV infection. These results show that a highly pathogenic avian influenza virus can be attenuated by modulating the caspase cleavage sites of NP and M2 and that these sites should be considered as novel targets for the design of live vaccines.