Impact of Specific Pattern Mutations on Plaque Formation and Attenuation Properties of Dengue Virus Serotype 3

Dengue fever is a major public health problem in several countries, including Thailand. The development of an effective dengue vaccine against all four serotypes is important for controlling the disease. An attenuated dengue-3 vaccine candidate, namely, DENV3-MBU-1, was derived from full-length cDNA-assembled segments based on the sequence of a prototype DENV3 vaccine candidate originating from the DENV3 strain 16562, which was passaged 30 times in primary green monkey kidney cells and four times in primary dog kidney cells. Compared to the parental DENV3-16562 sequence, this DENV3-MBU-1 contains seven nonsynonymous mutations in the premembrane (prM), envelope (E), and nonstructural proteins (NS2A, NS3, NS4). In this study, we aimed to characterize the mutations contributing to attenuation by generating different DENV3 mutants and evaluating their properties, including plaque formation and neurovirulence in mice. The DENV3-MBU-1 mutants, in which the NS proteins were reverted to those of the parental virus, exhibited an altered phenotype, manifested by changes in plaque size and virulence, resembling that of the parental virus. This suggests the important role of the NS segment in attenuation. Interestingly, the mutants of DENV3-MBU-1, in which only the structural proteins, but not the NS proteins were altered, showed changes in phenotype toward increased plaque size and neurovirulence. This suggests that attenuation may be influenced by specific combinations of mutations rather than single-point mutations. These findings, along with the attenuated DENV3 viruses generated, provide useful information for the development of a dengue vaccine.