The Effects Of Zika Virus NS3, NS4A And NS5 Proteins On Neurodevelopment In Mice

Zika virus is a positive-stranded RNA virus in the flavivirus genus of the Flaviviridae family.The infection of Zika virus is associated with fetal microcephaly and adult Guillain-Barre syndrome,and there are no effective vaccines and targeting drugs worldwide.Zika virus genome encodes 3 structure proteins(C,prM and E)and 8 non-structure proteins or peptide(NS 1,NS2A,NS2B,NS3,NS4A,2K,NS4B,NS5).Studies at cell level suggested that NS4A can inhibit neurogenesis.NS3 and NS5 protein play important roles in the life cycle of Zika virus and are hotspots in antiviral researches.However,there are no in vivo studies that proved which protein plays the key role in the neural development.In this study,we used NS3,NS4A and NS5 proteins to determine the effects in neuronal development.The genome of Zika virus was sequenced by RACE and reverse transcription PCR to confirm the sequence of this subject.And then,the sequence of these proteins fused with Flag-tag was amplified and constructed in pCIG vector.These viral proteins were electroporated in the mouse brain at 13.5 embryonic days(E13.5)by in utero,and the pCIG empty vector was used as control.Immunohistochemistry was used to detect the expression of proteins by Flag-tag,in the cerebral cortex of E18.5.These results showed that virus proteins can over expressed successfully in vivo.The detection results showed that after the NS4A expression,most of the cells were distributed in the intermediate zone(IZ)below the cortical laminar,while the control and the cells after NS3 or NS5 expression were mainly distributed in the cortical II-IV laminar layer.The ratio of CUX1 and TBR1 indicated that NS5 protein can change the cell fate.While the detection of PAX6 and NeuroD2 showed that no protein obstruct the neural precursor cell's differentiation,and Furthermore,we found that only NS5 can activate the Caspase3 pathway.This study found that both Zika virus NS3 and NS4A proteins can affect the migration of neurons in the mouse cerebral cortex,while the impact of NS4A was more obvious.NS5 may activate the Caspase3 signaling pathway to induce cell apoptosis and change the cell fate.For a conclusion,these 3 viral proteins play a role in the pathogenesis of fetal microcephaly in different mechanisms.