Molecular Analysis of Yellow Fever Virus Variants With Altered Phenotypes.

This study investigated the determinants of yellow fever (YF) virus pathogenesis at the molecular level by generating nucleotide and deduced amino acid sequences for the structural protein genes of attenuated variants of YF virus. In total, approximately 75 kilobases of nucleotide sequence were generated. The membrane (M) or envelope (E) protein gene sequences of four 17D-204 vaccine viruses and three French neurotropic vaccine viruses were determined and compared with published sequence data. Despite completely different routes of attenuation, the two vaccine strains shared a substitution at amino acid 35 in the M protein (M-35), and exhibited a cluster of substitutions in the E protein. Sequence analysis of a YF virus attenuated by limited passage in HeLa cells revealed that this virus consisted of a mixed population of variants. The structural proteins and two non-structural proteins were sequenced for several clones and the E protein was shown to have the highest rate of mutation. Comparison with the YF vaccine viruses suggested that M-67 and E-27 amino acid substitutions were important for the attenuated phenotype. A number of YF variant viruses were studied in which the E proteins had previously been demonstrated to have unusual antigenic profiles. Several of the variant viruses were shown to have altered virulence phenotypes in mice, including avirulence, intermediate virulence, persistence and complete lethality. The pathogenesis of these viruses in the murine brain was investigated further by determining the levels of infectious virus or viral RNA. Sequence analyses of the E protein genes of these viruses implicated E-153, E-240, E-305 and E-325 in the altered phenotypes. Overall, the results indicate that the M and E proteins are major molecular determinants of YF virus pathogenesis