A single nonsynonymous mutation on gene encoding E protein of Zika virus leads to increased neurovirulence in vivo

Zika virus can infect a wide range of tissues including the developmental brain of human fetuses, causing from mild to severe clinical diseases. Whether its genetic characteristics impacts on viral pathogenesis is incompletely understood. We have obtained viral variants through serially passage of a clinical Zika virus isolate (SW01) in neonatal mice in vivo and found some of which exhibited markedly increased virulence and neurotropism. By deep sequencing analysis, the more pathogenic viral variants were found to contain four dominant nonsynonymous nucleotide mutations on genes encoding E and NS2A proteins. Further investigation using molecularly cloned viruses revealed that a single 67D (Aspatic acid) to N (Asparagine) substitution on E protein is sufficient to confer the increased virulence and neurotropism. These findings provide new insight into Zika virus pathogenesis and suggest novel targets for the development of therapeutics. Author SummaryRecent large outbreaks of Zika virus infection worldwide have revealed an association between the viral infection and increased cases of specific neurological problems including Congenital Zika Syndrome (including microcephaly) and adult Guillain-Barre Syndrome. However, the determinants of the increased neurovirulence of Zika virus remain uncertain. One hypothesis is that some unique changes across the Zika viral genome have led to the occurrence of these neurological diseases. To test this hypothesis, we continuously propagated a clinical isolate of contemporary Zika virus (SW01) in neonatal mice brain for 11 times to obtain an mouse central nervous system (CNS) adapted Zika virus (MA-SW01) that showed significantly increased neurovirulence in vivo. We then discovered that a single G to A nucleotide substitution at the 1069 site of Zika virus open reading frame leading to a D (aspartic acid) to N (asparagine) in viral Envelope protein is responsible for the increased neurovirulence. These findings improve our understanding of the neurological pathogenesis of Zika virus and provide clues for the development of antiviral strategy.