Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells.

Mutations which increase the replication of dengue viruses in cell culture would greatly facilitate the manufacture of both a live attenuated or inactivated dengue virus vaccine. We have identified eight missense mutations in dengue virus type 4 (DEN4) that increase the plaque size and kinetics of replication of recombinant DEN4 virus in Vero cells. DEN4 viruses bearing these Vero cell adaptation mutations were also evaluated for the level of replication in the brains of mice. Two of these eight recombinant viruses expressing distinct mutations in NS3 were both restricted in replication in the brains of suckling mice. In contrast, six recombinant viruses, each encoding individual mutations in NS4B (five) or in NS5 (one), were not attenuated in mouse brain. Recombinant viruses encoding various combinations of these Vero cell adaptation mutations did not demonstrate enhanced replication in Vero cells over that exhibited by the single mutations. Finally, addition of a subset of the above non-attenuating, adaptation mutations to a DEN2/4 chimeric vaccine candidate was found to increase the virus yield in Vero cells by up to 500-fold. The importance of these Vero cell adaptation mutations in flavivirus vaccine design and development is discussed.