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A single mutation in the prM protein of Zika virus contributes to fetal microcephaly

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Science  28 Sep 2017:
eaam7120
DOI: 10.1126/science.aam7120
  • Fig. 1 Neurovirulence phenotypes of the contemporary ZIKV strains and its ancestral Asian strain.

    (A) Neurovirulence tests of different ZIKV strains in neonatal mice. P1 BALB/c mice were IC injected with 10 PFU of virus and mortality was observed for 26 days. CAM/2010: n = 24, VEN/2016: n = 21, SAM/2016: n = 22. MTQ/2015: n = 23. Log-rank test was performed for statistical analysis. ****p < 0.0001. (B to D) Littermate embryonic brains were injected with CAM/2010, VEN/2016 or culture media containing 2% FBS (Mock) at embryonic day 13.5 (E13.5) and inspected at E16.5 or E18.5. (B) Images of brains (E18.5). Red/yellow bars represent brain width/cerebral cortex length. (C) Nissl staining of E18.5 brains. CP: cortical plate, SP: subplate, IZ: intermediate zone, VZ: ventricle zone, SVZ: sub-ventricle zone. (D) Images of E16.5 cortices stained with phospho-Histone H3 (P-H3, red). Scale bars: 5 mm (B), 100 μm (C), 40 μm (D).

  • Fig. 2 Phylogenetic and molecular clock analysis of ZIKV strains of the Asian lineage.

    (A) The root-to-tip analysis using TempEst v1.5. The input was a Maximum Likelihood tree estimated using RAxML with 1000 bootstrap replicates (fig. S4). (B) The Bayesian phylogenetic tree estimated using BEAST v1.8.4. The positions of CAM/2010, VEN/2016, SAM/2016 and MTQ/2015 were indicated with black and red arrows. Conserved amino acid changes were inferred using the CAM/2010 strain as the parental strain. The green bars indicated the 95% highest probability density intervals of the age of the lineage. The details of the tree are shown in fig. S5.

  • Fig. 3 The S139N mutant virus showed enhanced neurovirulence in neonatal mice and virial replication in hNPCs.

    (A) Comparison of neurovirulence of ZIKV mutants in neonatal mice. 10 PFU of ZIKV was injected into the brains of P1 BALB/c mice. WT: n = 24; T106A: n = 22; S109N: n = 22; N130S: n = 23; S139N: n = 22; K709R: n = 23; A982V: n = 21; N3144S: n = 15. (B) 10 PFU of ZIKV VEN/2016 strain or the N139S reverse-mutant was injected into the brains of P1 BALB/c mice and the mortality were observed for 25 days. VEN/2016, n = 12; VEN/2016-N139S, n = 12. Log-rank test was performed for statistical analysis. ****p < 0.0001. (C) hNPCs were infected or mock infected with WT or S139N mutant. The virus titers in the culture supernatants were inspected by plaque assay at 72 hours post infection. **p < 0.01. (D) At 56 hours post infection, hNPCs were fixed and stained for ZIKV (green), the activated form of Caspase 3 (Cas3, red) and DAPI (gray). Scale bar: 40 μm.

  • Fig. 4 The S139N mutant causes more significant microcephaly.

    Littermate embryonic brains were injected with culture media (mock), WT or S139N mutant virus at E13.5, and inspected at E18.5. (A) Images of microcephalic brains (E18.5). Red/yellow bars represent brain width/cerebral cortex length. (B) Nissl staining of E18.5 brain cortices. (C) S139N mutant causes more apoptosis at E18.5. Images of cortices stained with Cas3 (gray), ZIKV (Green) and DAPI. Lower panel: quantification of Cas3+ cells. Mock: n = 9/7, WT: n = 9/6, S139N: n = 10/7. (D) Images of E18.5 brain cortices stained with P-H3 (Red) and ZIKV (Green) antisera. Lower panel: Quantification of P-H3+ cells in the VZ. Mock and WT: n = 9/4, S139N: n = 13/5. All data are means ± SD, t test. **p < 0.01, ***p < 0.001, ##p < 0.01, ###p < 0.001. n = slice numbers/brain numbers. Scale bars: 2 mm (A), 100 μm (B), 40 μm (C, D).

Supplementary Materials

  • A single mutation in the prM protein of Zika virus contributes to fetal microcephaly

    Ling Yuan, Xing-Yao Huang, Zhong-Yu Liu, Feng Zhang, Xing-Liang Zhu, Jiu-Yang Yu, Xue Ji, Yan-Peng Xu, Guanghui Li, Cui Li, Hong-Jiang Wang, Yong-Qiang Deng, Menghua Wu, Meng-Li Cheng, Qing Ye, Dong-Yang Xie, Xiao-Feng Li, Xiangxi Wang, Weifeng Shi, Baoyang Hu, Pei-Yong Shi, Zhiheng Xu, Cheng-Feng Qin

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S14
    • Tables S1 and S2
    • References