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

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Science  17 Nov 2017:
Vol. 358, Issue 6365, pp. 933-936
DOI: 10.1126/science.aam7120
  • Fig. 1 Neurovirulence phenotypes of the contemporary ZIKV strains and their ancestral Asian strain.

    (A) Neurovirulence tests of different ZIKV strains in neonatal mice. P1 BALB/c mice were intracerebrally injected with 10 plaque-forming units (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 (n, number of mice). 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% fetal bovine serum (mock) at E13.5 and inspected at E16.5 or E18.5. (B) Images of brains (E18.5). Red and yellow bars represent brain width and cerebral cortex length, respectively. (C) Nissl staining of E18.5 brains. CP, cortical plate; SP, subplate; IZ, intermediate zone; VZ, ventricle zone; SVZ, subventricle zone. (D) Images of E16.5 cortices stained with phosphorylated 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) Root-to-tip analysis using TempEst v1.5. The input was a maximum likelihood tree estimated using RAxML (Randomized Axelerated Maximum Likelihood) with 1000 bootstrap replicates (fig. S6). R2, coefficient of determination. (B) Bayesian phylogenetic tree estimated using BEAST v1.8.4. The positions of CAM/2010, VEN/2016, SAM/2016, and MTQ/2015 are indicated with black (CAM/2010) and red (VEN/2016, SAM/2016, and MTQ/2015) arrows. Conserved amino acid changes were inferred using the CAM/2010 strain as the parental strain. The green bars indicate the 95% highest probability density intervals of the age of the lineage. The details of the tree are shown in fig. S7. V, Val; A, Ala; N, Asn; S, Ser; M, Met; L, Leu; P, Pro; T, Thr.

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

    (A) Comparison of neurovirulence of ZIKV mutants in neonatal mice. 10 PFU of ZIKV were 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 (n, number of mice). (B) 10 PFU of ZIKV VEN/2016 strain or the N139S reverse mutant were injected into the brains of P1 BALB/c mice, and mortality was observed for 25 days. VEN/2016: n = 12; VEN/2016-N139S: n = 12. A log-rank test was performed for statistical analysis. ****P < 0.0001. (C) hNPCs were infected or mock-infected with WT or S139N mutant. Virus titers in the culture supernatants were inspected by plaque assay at 72 hours postinfection. **P < 0.01. Error bars indicate means ± SD. (D) At 56 hours postinfection, hNPCs were fixed and stained for ZIKV (green), the activated form of caspase 3 (Cas3, red), and 4′,6-diamidino-2-phenylindole (DAPI) (gray). Scale bar: 40 μm.

  • Fig. 4 The S139N mutant causes more severe 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 and yellow bars represent brain width and cerebral cortex length, respectively. (B) Nissl staining of E18.5 brain cortices. (C) The S139N mutant causes more apoptosis at E18.5. (Top) Images of cortices stained with Cas3 (gray), ZIKV (green), and DAPI (blue). (Bottom) Quantification of Cas3+ cells. Mock: n = 9/7; WT: n = 9/6; S139N: n = 10/7 (n, slice numbers/brain numbers). (D) (Top) Images of E18.5 brain cortices stained with P-H3 (red) and ZIKV (green) antisera. (Bottom) Quantification of P-H3+ cells in the VZ. Mock and WT: n = 9/4; S139N: n = 13/5. All data are means ± SD (error bars); Student’s t test. **P < 0.01; ***P < 0.001; ##P < 0.01; ###P < 0.001. Scale bars: 2 mm (A), 100 μm (B), 40 μm (C and 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

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