Investigating the role of non-structural genes in the genotype and phenotype of yellow fever vaccine strain 17D

The first yellow fever outbreak was recorded in 1648 in the Yucatan Peninsula. Almost 300 years later, a live-attenuated vaccine against yellow fever virus, the causative agent of yellow fever, was successfully derived by serial passage of Asibi virus 176 times in chick and mouse embryo tissue. The resulting vaccine strain, 17D, gave rise to all substrains in use today. Strain 17D and the resulting substrains, 17DD and 17D-204, were found to differ from Asibi virus by 20 amino acid substitutions with nine substitutions in the structural proteins of the virus and the remaining eleven in the non-structural proteins. Despite the long-term use and success of the 17D vaccine, it remains unknown which of the 20 residues contribute to its stably attenuated phenotype. By utilizing previously established genotypic and phenotypic differences between 17D and Asibi viruses, this dissertation aims to determine which NS proteins contribute to these differences. Using an infectious clone system, non-structural substitutions from 17D-204 virus were introduced into an Asibi infectious clone while Asibi virus residues were introduced into a 17D-204 infectious clone. The impact of the introduced substitutions on genetic diversity, multiplication kinetics, sensitivity to the antiviral Ribavirin, and attenuation in an AG129 mouse model were evaluated. 17D-204 virus is known to have reduced genetic diversity, increased multiplication kinetics in A549 cells, increased resistance to Ribavirin, and is attenuated in AG129 mice when compared to Asibi virus. These studies identified substitutions in several non-structural proteins that significantly impact genetic diversity (NS2B, NS4B, and NS5) and multiplication kinetics (NS5), providing evidence for the involvement of the NS proteins in 17D-204 viral attenuation. Substitutions within these proteins warrant further study. It was also found that the lack of genetic diversity exhibited by vaccine strains of yellow fever virus is not universal to attenuated strains of the virus. The attenuated FVV HeLa p5 strain was found to have similar genetic diversity and sensitivity to Ribavirin when compared with the original unpassaged strain, FVV. These findings highlight the importance of 17D-204 virus’s genetic stability and the contribution of that stability to the strain’s continued use as a live attenuated vaccine.