Two genetic differences between closely-related Zika virus strains determine pathogenic outcome in mice

Recent Zika virus (ZIKV) outbreaks and unexpected clinical manifestations of ZIKV infection have prompted an increase in ZIKV-related research. Here we identify two strain-specific determinants of ZIKV virulence in mice. We found that H/PF/2013 caused 100% lethality in Ifnar1-/- mice, whereas PRVABC59 caused no lethality; both strains caused 100% lethality in Ifnar1-/-Ifngr1-/- DKO mice. Deep sequencing revealed a high-frequency variant in PRVABC59 not present in H/PF/2013: a G to T change at nucleotide 1965 producing a Val to Leu substitution at position 330 of the viral envelope (E) protein. We show that the V330 variant is lethal on both strains, whereas the L330 variant is attenuating only on the PRVABC59 background. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain, but not H/PF/2013. The consensus sequences of H/PF/2013 and PRVABC59 differ by 3 amino acids, but these were not responsible for the difference in virulence between the two strains. H/PF/2013 and PRVABC59 differ by an additional 31 non-coding or silent nucleotide changes. We made a panel of chimeric viruses with identical amino acid sequences but nucleotide sequences derived from H/PF/2013 or PRVABC59. We found that 6 nucleotide differences in the 3' quarter of the H/PF/2013 genome were sufficient to confer virulence in Ifnar1-/- mice. Altogether, our work identifies 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).IMPORTANCE Contemporary ZIKV strains are closely related and often used interchangeably in laboratory research. Here we identify two strain-specific determinants of ZIKV virulence which are evident only in Ifnar1-/- mice, not Ifnar1-/-Ifngr1-/- DKO mice. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain, but not H/PF/2013. We further identify a second virulence determinant in the H/PF/2013 strain, which is driven by the viral nucleotide sequence, not the amino acid sequence. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used strains of ZIKV, in two widely used mouse models of ZIKV pathogenesis. Our results highlight that even very closely related virus strains can produce significantly different pathogenic phenotypes in common laboratory models.