Molecular determinants of Yellow Fever Virus pathogenicity in Syrian Golden Hamsters: one mutation away from virulence

Yellow fever virus (Flavivirus genus) is an arthropod-borne pathogen, which can infect humans, causing a severe viscerotropic disease with a high mortality rate. Adapted viral strains allow the reproduction of yellow fever disease in hamsters with features similar to the human disease. Here, we used the Infectious Subgenomic Amplicons reverse genetics method to produce an equivalent to the hamster-virulent strain, Yellow Fever Ap7, by introducing a set of four synonymous and six nonsynonymous mutations into a single subgenomic amplicon, derived from the sequence of the Asibi strain. The resulting strain, Yellow Fever Ap7M, induced a disease similar to that described for Ap7 in terms of symptoms, weight evolution, viral loads in the liver and lethality. Using the same methodology, we produced mutant strains derived from either Ap7M or Asibi viruses and investigated the role of each of Ap7M nonsynonymous mutations in its in vivo phenotype. This allowed identifying key components of the virulence mechanism in hamsters. In Ap7M virus, the reversion of either E/Q27H or E/D155A mutations led to an important reduction of both virulence and in vivo replicative fitness. In addition, the introduction of the single D155A Ap7M mutation within the E protein of the Asibi virus was sufficient to drastically modify its phenotype in hamsters toward both a greater replication efficiency and virulence. Finally, inspection of the Asibi strain E protein structure combined to in vivo testing revealed the importance of an exposed a-helix in domain I, containing residues 154 and 155, for Ap7M virulence in hamsters.