VIRAL GENETIC DETERMINANTS OF ZIKA VIRUS PATHOGENESIS

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus primarily transmitted by mosquitoes and ticks. Recent ZIKV outbreaks have produced serious human disease, including neurodevelopmental malformations and Guillain-Barré syndrome. These outcomes were not associated with ZIKV infection prior to 2013, raising the possibility that viral genetic changes could contribute to new clinical manifestations. In my studies, I investigated the role of E protein glycosylation in ZIKV pathogenesis. I found that glycosylated viruses were highly pathogenic in Ifnar1-/- mice, whereas non-glycosylated viruses were attenuated, producing lower viral loads in the serum and brain when inoculated subcutaneously, but replicating equally well in the brain when inoculated intracranially. These results suggest that E glycosylation is advantageous in the periphery but not within the brain. Accordingly, I found that glycosylation facilitated infection of lectin expressing cells, possibly explaining the attenuation of non-glycosylated ZIKV in mice. Additionally, I found that adding a glycan back to the E protein at a different position (N67) does not functionally complement the loss of the glycan at position N154. I also discovered two strain-specific determinants of ZIKV virulence in mice. I found that H/PF/2013 caused 100% lethality in Ifnar1-/- mice, whereas PRVABC59 caused no lethality. Deep sequencing revealed a high-frequency variant (V330L) in PRVABC59 not present in H/PF/2013. I showed that the V330 variant is lethal on both strains, whereas the L330 variant is attenuating only on the PRVABC59 background. To investigate the remaining differences between the two strains, I made a panel of chimeric viruses with nucleotide sequences derived from H/PF/2013 or PRVABC59. I found that 6 nucleotide differences in the 3’ quarter of the H/PF/2013 genome were sufficient to confer virulence in Ifnar1-/- mice. Altogether, I confirmed that the E protein glycosylation mediates pathogenesis of ZIKV from both Asian and African-lineage strains, and I identified a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis (Ifnar1-/- and Ifnar1-/- Ifngr1-/- DKO mice). My studies emphasize how small genetic changes in viruses can lead to drastically different pathogenic phenotypes in common laboratory models.Doctor of Philosoph