Defining molecular determinants of attenuation and virulence for yellow fever virus

This dissertation investigated the molecular determinants of attenuation and virulence for yellow fever virus (YFV) using the mouse model of disease. Despite the availability of a live, attenuated vaccine for YFV since 1937 (17D), little is still known about the molecular basis for attenuation and virulence for YFV. Based on genomic sequence comparisons of differential YFV strains, three proteins (M, E, and NS4B) were chosen to investigate their contribution to attenuation and virulence of YFV. Substitutions in the M, E, and NS4B proteins were introduced into either the wild-type Asibi or attenuated 17D-204 infectious clones, recombinant viruses were recovered, and their phenotypes in cell culture and the mouse models of disease were investigated. No clear multiplication differences were observed in cell lines between either wild-type Asibi, attenuated 17D-204, or the mutant infectious clone-derived viruses. It was found that the wild-type Asibi virus could be attenuated for mouse neuroinvasive and/or neurovirulence phenotype by introducing 17D substitutions at residues M-36, E-305, E-325, E-380, and NS4B-95 into the Asibi infectious clone. The residues at E-305 and E-380 both appear to be major determinants of attenuation, as shown by attenuation of mouse neuroinvasive and neurovirulence phenotypes. However, when the 17D residue was introduced into the Asibi backbone at position E-299, the mouse neuroinvasive and neurovirulence phenotypes of Asibi virus were increased, rather than decreased. The mouse neuroinvasive phenotype of the 17D-204 vaccine virus could not be increased by introducing wild-type Asibi substitutions at residues M-36, E-299, E-305, E-323/E-325, E-380, and NS4B-98. Furthermore, concomitant introduction of E-299, E-305, E-325, and E-380 did not result in an increase in mouse neuroinvasive phenotype for 17D-204 infectious clone-derived virus. Interestingly, when the wild-type Asibi residue was introduced into the 17D-204 backbone at position NS4B-95, the mouse neuroinvasive phenotype of 17D-204 was increased up to 160-fold. Substitutions at E-299, E-305, and E-380 in the 17D-204 backbone were able to increase the mouse neurovirulence phenotype of 17D-204.; Overall, these results indicate that the attenuation of 17D-204 vaccine virus is multi-factorial, and this may explain the excellent safety record of the 17D vaccine with very few examples of reversion to virulence.