Biologic effects of introducing additional basic amino acid residues into the hemagglutinin cleavage site of a virulent avian influenza virus.

We mutated the virulent avian influenza virus A/turkey/Ontario/7732/66 (H5N9)[Q-R-R-R-K-K-R\G at the hemagglutinin (HA) cleavage site] to create a mutant, R(MO-0), with additional basic residues at this site (Q-R-R-R-R-R-K-K-R\G) by reverse genetics. When tested in chicken embryo fibroblast culture, this mutant showed reduced HA cleavability compared to that of the wild-type virus, but its plaque size was not appreciably altered. Virulence of the R(MO-0) virus in chickens was lower than that of the wild-type virus. These findings indicate that addition of excessive basic residues to an optimal recognition sequence for HA cleavage enzymes at the cleavage site is deleterious for HA cleavability. Previously, we showed that a mutant containing the suboptimal HA cleavage site sequence for cleavage enzyme recognition also had reduced HA cleavability and virulence compared to the wild-type virus. We conclude that the data presented here further substantiate our belief that the level of HA cleavability correlates with the degree of virulence when all other genetic characteristics are considered equal, irrespective of the mechanisms by which HA cleavability is reduced.