Investigation of the interplay of yellow fever virus structural protein epitopes and genetic diversity
Yellow fever virus (YFV), a mosquito-borne flavivirus, is responsible for the disease yellow fever (YF), which is characterized by hemorrhagic fever and multiorgan failure. The disease is prevented by a live attenuated vaccine (LAV), strain 17D, that was derived from serial passage of the wild type (WT) strain Asibi in chicken tissue. The mechanism of attenuation of 17D is incompletely understood. This dissertation investigates the contribution of the envelope (E) protein in YFV attenuation through its role in genetic diversity, tissue tropism and recognition by WT and vaccine specific antibodies that bind E protein. Using infectious clones (i.c.) of both Asibi and 17D viruses, structural chimeras with swapped prME, E protein domain III (EDIII) and single site mutations at each of the residues that differ between WT and vaccine strain in structural proteins were generated. Using these chimeras, the determinants of focus morphology were mapped to residues E-52 and E-380. E-305 was shown to be critical to the increased multiplication kinetics of 17D virus compared to Asibi. Genotypic stability was investigated using Illumina deep sequencing methods and it was shown that the E protein contributes to the differences seen in the genotype of Asibi and 17D viruses, whereas M protein does not. Despite this, prME was not shown to contribute to YFV susceptibility to the antiviral Ribavirin. Tissue tropism was correlated with WT and vaccine epitopes as residues in EDI were critical to viscerotropism and WT mAb recognition and residues within EDIII were shown to be critical to neurotropism and vaccine mAb recognition. It was found that the attenuating processes of 17D were not comparable to the JEV SA14-14-2 LAV. This is unsurprising due to the empirical nature of legacy LAVs and suggests there are many mechanisms that could be employed to generate future flavivirus LAVs. Overall, the role of E in the attenuation of 17D seems to rely on several critical residues (E-52, E-170, E-305, E-325 and E-380) many of which contribute the net positive charge of the 17D virion.