Key Residue in the Precursor Region of M Protein Contributes to the Neurovirulence and Neuroinvasiveness of the African Lineage of Zika Virus.

The Zika virus (ZIKV) represents an important global health threat due to its unusual association with congenital Zika syndrome. ZIKV strains are phylogenetically grouped into the African and Asian lineages. However, the viral determinants underlying the phenotypic differences between the lineages remain unknown. Here, multiple sequence alignment revealed a highly conserved residue at position 21 of the premembrane (prM) protein, which is glutamic acid and lysine in the Asian and African lineages, respectively. Using reverse genetics, we generated a recombinant virus carrying an E21K mutation based on the genomic backbone of the Asian lineage strain FSS13025 (termed E21K). The E21K mutation significantly increased viral replication in multiple neural cell lines with a higher ratio of M to prM production. Animal studies showed E21K exhibited increased neurovirulence in suckling mice, leading to more severe defects in mouse brains by causing more neural cell death and destruction of hippocampus integrity. Moreover, the E21K substitution enhanced neuroinvasiveness in interferon alpha/beta (IFN-α/β) receptor knockout mice, as indicated by the increased mortality, and enhanced replication in mouse brains. The global transcriptional analysis showed E21K infection profoundly altered neuron development networks and induced stronger antiviral immune response than wild type (WT) in both neural cells and mouse brains. More importantly, the reverse K21E mutation based on the genomic backbone of the African strain MR766 caused less mouse neurovirulence. Overall, our findings support the 21st residue of prM functions as a determinant for neurovirulence and neuroinvasiveness of the African lineage of ZIKV.