Role of nonstructural protein in limited genetic diversity of yellow fever 17D vaccine virus

ABSTRACT Many successful live attenuated vaccines (LAVs) were derived empirically and little is known about their mechanisms of attenuation. The best characterized of these LAVs is poliovirus where attenuation has been mapped to the 5'NCR and capsid genes. Increased understanding the mechanism of attenuation of licensed LAVs will help in the rational development of future LAVs. The disease yellow fever is controlled by the use of a live attenuated vaccine, strain 17D, derived from wild-type (WT) strain Asibi, and differ by 20 amino acids; 9 in the structural genes and 11 in the nonstructural (NS) genes. Our overall goal is to understand the mechanism of attenuation of 17D vaccine, which is poorly understood. Next Generation Sequencing (NGS) technology has great applications to vaccine development and quality control and safety of LAVs. We have compared WT Asibi and 17D vaccine by NGS and found that Asibi is a typical RNA virus with a quasispecies population while, surprisingly, 17D vaccine has very little evidence of quasispecies, and we believe that this may contribute to the attenuated phenotype of the vaccine virus. We have shown that the restricted quasispecies in 17D vaccine virus is due to mutation(s) in the NS proteins of the replication complex as a whole, rather than the RNA dependent RNA polymerase (RdRp) alone. In addition, ribavirin is an antiviral drug that introduces mutations into RNA genomes during replication due to the lack of fidelity of the viral RdRp. We have shown that WT Asibi virus is sensitive to ribavirin while 17D vaccine virus is relatively resistant suggesting that a high fidelity replication complex potentially contributes to the attenuated phenotype of 17D vaccine. We believe that investigation of the mechanism of limited quasispecies in 17D vaccine virus may have important applications to understanding the molecular basis of attenuation of 17D vaccine, other LAVs, and development of future flavivirus LAVs. In this application we will identify the viral genes that contribute to limited quasispecies of 17D vaccine. We hypothesize that the restricted quasispecies in 17D vaccine virus is due to multiple mutations in the NS proteins of the replication complex as a whole, rather than the RdRp alone, encodes an attenuated phenotype, and multiple mutations contribute to the very low rate of reversion to virulence. The objective of this proposal is to perform targeted studies to identify which YFV NS genes contribute to the restricted quasispecies of 17D vaccine to propose a hypothesis to investigate the role of NS genes in the mechanism of attenuation of 17D vaccine. This will be achieved via three specific aims: Aim 1 will investigate the quasispecies population and ribavirin sensitivity of Asibi/17D mutants to identify residues in NS genes that contribute to restricted quasispecies population and ribavirin resistance of 17D vaccine; Aim 2 will generate Asibi mutants that are resistant to ribavirin, identify the nucleotide changes responsible for resistance, and characterize the quasispecies population of the mutants; and Aim 3 will investigate the phenotype of Asibi/17D mutants in AG129 mice to determine if ribavirin resistance and/or lack of quasispecies correlates with an attenuated phenotype.