Pattnaik AK, Xiang SH, Osorio FA, Petro TM, Delhon G, Vu HLX, Natarajan SK, Pattnaik A, Annamalai AS, Muthukrishnan E, Steffen D, Sahoo BR
Zika virus (ZIKV), a mosquito-transmitted flavivirus, responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, re-emerged in the last decade causing serious human diseases including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently outstanding. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic, being absent in many of the African isolates while present in all isolates from the recent outbreaks. In the present study, we interrogated the role of this sequence motif and glycosylation of the E protein in pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or from which N-linked glycosylation site is mutated by single amino acid substitution, are highly attenuated and non-lethal. The mutant viruses replicate poorly in the brain of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE Recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for their ability to cause mortality in mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein as an important factor contributing to ZIKV pathogenicity.