Contributions of Structural and Non-structural Genes to the Attenuation of the 17D Vaccine

Live-attenuated 17D vaccine, developed to control Yellow Fever virus (YFV), was derived by serial passage of wild-type Asibi strain 176 times in mouse and chicken tissues. Despite the derivation of 17D over 75 years ago, the mechanism of attenuation is still poorly understood. The genomes of wild-type Asibi virus and the 17D vaccine substrains 17D-204, 17D-213 and 17DD were sequenced using Sanger sequencing and shown to differ from wild-type Asibi virus by 9 common amino acid substitutions in the structural protein genes, 11 common amino acid substitutions in the non-structural (NS) protein genes, and four nucleotide changes in the 3’ non-coding region. The NS proteins are required for viral replication and modulate the host immune response. The hypothesis to be tested in this dissertation is to determine whether or not structural and non-structural (NS) protein genes encode major genetic determinants of attenuation and contribute to decreased virulence of the 17D vaccines. RNA viruses are known to exist as populations of genetically related virions, known as quasispecies population, which contributes to the fitness and virulence of a virus. Infectious clone (IC)-derived chimeric viruses will be used to investigate contributions of NS protein genes to the generation of quasispecies population by assessing Shannon entropy of nucleotide diversity and frequency of RNA subpopulation in Specific Aim 1. Specific Aim 2, determines if the genetic determinants in NS protein genes that influence quasispecies, also influence multiplication kinetics. Finally, induced cytokines and gene expression of IC-derived viruses will be determined to assess whether or not attenuating mutations in NS proteins abate immune regulation typical of wild-type infections in Specific Aim 3. The results of this study showed that the attenuated genotype and phenotype of 17D vaccine is multi-genic, requiring both structural and NS protein genes.