Research ArticleVIROLOGY

Congenital Zika syndrome is associated with maternal protein malnutrition

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Science Advances  10 Jan 2020:
Vol. 6, no. 2, eaaw6284
DOI: 10.1126/sciadv.aaw6284
  • Fig. 1 Protein malnutrition reduces maternal body weight and enhances ZIKV load.

    (A) Relationship between undernutrition, measured as the number of hospitalized cases due to any undernutrition, and microcephaly cases that were confirmed or under investigation in Brazil since the first American ZIKV outbreak. Taking into consideration the data of 24 tropical states, a significant positive correlation was found between these variables (Pearson correlation: *r = 0.4, P-adj = 0.028). Each symbol represents a Brazilian tropical state. Geographical regions to which these states belong are indicated. hab, inhabitants. (B) Distribution of daily protein consumption of interviewed mothers with CZS children. Those that were under the recommended amount of protein intake are in gray, and those who achieved values above recommendations are in black. Those mothers considered under protein restriction consumed 53.86 ± 5.49 g/day on average. Red lines indicate the daily average protein intake for the Northeast region (solid line) and particularly for the state of Ceará (dashed line). Mothers from Ceará have a mean protein intake significantly larger than CZS interviewed mothers (t = −7.5, P < 0.001). (C) Schematic description of experimental groups and infection protocol. Wild-type dams were divided into control (Co) diet and low-protein diet (LP), and at E12, they were intraperitoneally injected either with 106 PFU of a Brazilian ZIKV strain from a stock with 3.4 × 106 PFU/ml (injected volume, 295 μl) or with the supernatant of Aedes albopictus C6/36 cells (Mock). P0, postnatal day 0. (D) Quantification of maternal body weight during pregnancy (n of dams per group: Co/Mock, 8; Co/ZIKV, 6; LP/Mock, 8; and LP/ZIKV, 11). Differences in maternal weight were only registered at E15 [Kruskal-Wallis, 11.375 (**)] [multiple comparisons with Mann-Whitney U: Co/Mock versus Co/ZIKV, Z = 0.000, Co/Mock versus LP/Mock, Z = −1.892; Co/Mock versus LP/ZIKV, Z = −2.522 (**); Co/ZIKV versus LP/Mock, Z = −2.027 (*); Co/ZIKV versus LP/ZIKV, Z = −2.720 (**); and LP/Mock versus LP/ZIKV, Z = −0.496]. Total leptin (E) and insulin-like growth factor 1 (IGF1) (F) maternal serum levels were measured by specific murine enzyme-linked immunosorbent assay (ELISA) at E15. The levels of both hormones were significantly lower only in LP groups than in Co diet groups [n per group: Co/Mock = 4; Co/ZIKV = 4 (leptin)/3 (IGF1); LP/Mock = 4; and LP/ZIKV = 3] [Kruskal-Wallis (leptin), 10.617 (*); multiple comparisons with Mann-Whitney U: Co/Mock versus LP/Mock, Z = −2.309 (*); Co/Mock versus LP/ZIKV, Z = −2.121 (*); Co/ZIKV versus LP/Mock, Z = −2.309 (*); and Co/ZIKV versus LP/ZIKV, Z = −2.121 (*)] [Kruskal-Wallis (IGF1), 9.967 (*); multiple comparisons with Mann-Whitney U: Co/Mock versus LP/Mock, Z = −2.309 (*); Co/Mock versus LP/ZIKV, Z = −2.121 (*); Co/ZIKV versus LP/Mock, Z = −2.121 (*); and Co/ZIKV versus LP/ZIKV, Z = −1.964 (*)]. The error bar represents mean ± SD. (G) Viral load was quantified by RT-qPCR on E15 maternal spleen and expressed as ZIKV RNA copies per gram (n of dams per group: Mock = 4; Co/ZIKV = 4; and LP/ZIKV = 5). High levels of ZIKV RNA were found in most of LP/ZIKV spleen tissues [Mann-Whitney U: Co/ZIKV versus LP/ZIKV, Z = −2.147 (*)]. *P < 0.05, **P < 0.01, and P > 0.05.

  • Fig. 2 Placental alterations 3 days after ZIKV infection in LP group.

    (A) Schematic representation of a placental structure. Normal labyrinth structure is characterized by separate maternal sinus with anucleated erythrocytes and embryonic capillaries with nucleated blood cells. (B) Viral load in the placentas was quantified by RT-qPCR (n per group E15: Mock = 13; Co/ZIKV = 10; and LP/ZIKV = 11; placentas were derived from five independent litters in each group). Lines indicate the geometric mean. Detectable viral RNA was found in 40% of Co/ZIKV placenta and in ~50% of LP/ZIKV placentas. No statistically significant difference in the amount of viral RNA was detected between means of Co/ZIKV and LP/ZIKV (P > 0.05). (C) Analysis of placental sections with hematoxylin and eosin (H&E) staining. Representation of altered labyrinth morphology with nucleated (embryonic, arrow) and anucleated (maternal, arrowheads) blood cells sharing the same vascular spaces found in LP/ZIKV placentas is shown. Scale bar, 20 μm. (D) Necrotic areas were identified in the spongiotrophoblast of some LP/ZIKV cases (top), with abundant examples of pyknosis and karyorrhexis, suggesting the occurrence of this degenerative process. In addition, an example of the hemorrhagic area found in the LP/ZIKV labyrinth is shown (bottom). White frames were used to indicate the location of the lesion. Scale bar, 50 μm.

  • Fig. 3 Maternal undernutrition associated with ZIKV during pregnancy results in neurodevelopmental impairments 3 days after infection (E15).

    (A) Quantification of body weight of embryos at E15 [Kruskal-Wallis, 8.360 (*); multiple comparisons with Mann-Whitney U: Co/Mock versus LP/ZIKV, Z = −1.959 (*)]. Variance is significantly different between groups, as estimated by Bartlett test K-squared, 19.929 (***). (B) Analysis of microglia/macrophage recruitment with Iba1+ immunostaining (red) reveals the presence of reactive cells in the developing brain of LP/ZIKV group, in particular, in the lateral ventricles. (C) Immunostaining and quantification of cycling cells in the proliferative zone show a reduction of Ki67+ cells (green) in LP/ZIKV cortex. Analysis of variance (ANOVA) F = 3.838 (*); least significant difference (LSD): Co/Mock versus LP/ZIKV (*) (P-adj > 0.05), Co/ZIKV versus LP/ZIKV (*) (P-adj > 0.05), LP/Mock versus LP/ZIKV (*) (P-adj > 0.05), df = 3. DAPI, 4′-6-diamidino-2-phenylindole. (D) Immunostaining and quantification of early-born neurons labeled with CTIP2+ (green). LP/ZIKV results in a reduction in cortical neurons compared with controls. ANOVA F = 4.77 (*); LSD: Co/ZIKV versus LP/ZIKV (*) (P-adj > 0.05), LP/Mock versus LP/ZIKV (**) [P-adj < 0.05 (**)], df = 3 (n = 3 per each group; in all cases, three sections of each brain were quantified and averaged). *P < 0.05 and **P < 0.01. The error bar represents mean ± SD. Scale bars, 50 μm.

  • Fig. 4 LP and congenital ZIKV infection induce gene expression deregulation in embryonic brains.

    (A) Pathways reflecting biological processes that are deregulated in the LP/ZIKV in comparison to Co/ZIKV after RNA-seq analyses. Only those with large significant differences are presented. (B) Circos plot showing the 60 hub genes, most (red) and least (blue) expressed in LP/ZIKV (in comparison to Co/ZIKV) shared between relevant cellular processes. Individual selected genes that are significantly down-regulated (C) or up-regulated (D) in LP/ZIKV in comparison to Co/ZIKV. FC, fold change.

  • Fig. 5 LP mediates brain size reduction after ZIKV.

    (A) Representative three-dimensional reconstructions of brain anatomy of neonates obtained from μCT illustrate size reduction in the LP/ZIKV group. Scale bar, 1 mm. (B) Coronal histological DAPI-stained sections also illustrate a cortical wall reduction (scale bar, 0.25 mm). The white measurement indicates the cortex thickness in the Co/Mock group, illustrated with the same size in all the other groups for comparative purposes. (C) Quantification of total body weight of newborns shows a significant decrease in the LP/ZIKV weight at P0. Kruskal-Wallis, 17.217 (***); multiple comparisons with Mann-Whitney U: Co/Mock versus Co/ZIKV, Z = −1.724; Co/Mock versus LP/Mock, Z = −1.328; Co/Mock versus LP/ZIKV, Z = −3.634 (***); Co/ZIKV versus LP/Mock, Z = −0.216; Co/ZIKV versus LP/ZIKV, Z = −2.621 (**); and LP/Mock versus LP/ZIKV, Z = −1.857. (D) Brain centroid size (CS), the square root of the summed squared distances of all digitized points to the centroid of the configuration, was obtained after the digitization of the three-dimensional position of 57 anatomical points. Kruskal-Wallis, 22.541 (***); multiple comparisons with Mann-Whitney U: Co/Mock versus Co/ZIKV, Z = −0.707; Co/Mock versus LP/Mock, Z = −1.768; Co/Mock versus LP/ZIKV, Z = −3.158 (**); Co/ZIKV versus LP/Mock, Z = −1.609; Co/ZIKV versus LP/ZIKV, Z = −2.982 (**); and LP/Mock versus LP/ZIKV, Z = −3.530 (***). (E) The proportion of cortex centroid size in relation to total brain centroid size. This proportion in LP/ZIKV group is smaller than in undernourished pups that have not been infected with ZIKV. Kruskal-Wallis, 13.743 (**); multiple comparisons with Mann-Whitney U: Co/Mock versus Co/ZIKV, Z = −0.354; Co/Mock versus LP/Mock, Z = −1.768; Co/Mock versus LP/ZIKV, Z = −2.216 (*); Co/ZIKV versus LP/Mock, Z = −2.082 (*); Co/ZIKV versus LP/ZIKV, Z = −1.765; and LP/Mock versus LP/ZIKV, Z = −3.043 (**). *P < 0.05, **P < 0.01, ***P < 0.001, and P > 0.05. The error bars represent means ± SD.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/2/eaaw6284/DC1

    Fig. S1. Distribution of simulated r coefficients for the correlation between undernutrition and microcephaly cases in tropical states of Brazil obtained after permutation of the original dataset.

    Fig. S2. Number of ZIKV infection confirmed cases in each Brazilian state.

    Fig. S3. Viral load in E18 placentas was quantified by RT-qPCR.

    Fig. S4. Confirmation of ZIKV detection by immunohistochemistry in E15.

    Fig. S5. Viral replication in LP/ZIKV placentas at E15.

    Fig. S6. H&E staining in E15 placentas.

    Fig. S7. Viral load in E15 and E18 embryonic brains quantified by RT-qPCR.

    Fig. S8. ZIKV-infected cells colocalize with endothelial cells in E15 brains.

    Fig. S9. ZIKV-infected cells colocalize with microglial cells in E15 brains.

    Fig. S10. Staining of J2 on E15 embryonic brains colocalized with IB4.

    Fig. S11. Network describing altered genes involved in the mTOR pathway.

    Fig. S12. Morphometric analysis of brain images obtained through μCT.

    Fig. S13. Skull size of LP/ZIKV newborns is reduced.

    Fig. S14. Summary of the results obtained with the experimental model.

    Fig. S15. Validation of differentially expressed genes by RT-qPCR in E15 brains of Co/ZIKV and LP/ZIKV.

    Table S1. Relationship between cases of undernutrition reported in hospitals and medical services and cases of microcephaly between 2015 and 2018.

    Table S2. The estimated amount of daily protein (g) intake for each of the 83 interviewed mothers who have children with CZS.

    Table S3. Relative risk of CZS in relation to protein intake.

    Table S4. Maternal body weight (mean and SD) at the different pregnancy stages of the mouse model.

    Table S5. Differentially expressed genes between Co/ZIKV and LP/ZIKV.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. Distribution of simulated r coefficients for the correlation between undernutrition and microcephaly cases in tropical states of Brazil obtained after permutation of the original dataset.
    • Fig. S2. Number of ZIKV infection confirmed cases in each Brazilian state.
    • Fig. S3. Viral load in E18 placentas was quantified by RT-qPCR.
    • Fig. S4. Confirmation of ZIKV detection by immunohistochemistry in E15.
    • Fig. S5. Viral replication in LP/ZIKV placentas at E15.
    • Fig. S6. H&E staining in E15 placentas.
    • Fig. S7. Viral load in E15 and E18 embryonic brains quantified by RT-qPCR.
    • Fig. S8. ZIKV-infected cells colocalize with endothelial cells in E15 brains.
    • Fig. S9. ZIKV-infected cells colocalize with microglial cells in E15 brains.
    • Fig. S10. Staining of J2 on E15 embryonic brains colocalized with IB4.
    • Fig. S11. Network describing altered genes involved in the mTOR pathway.
    • Fig. S12. Morphometric analysis of brain images obtained through μCT.
    • Fig. S13. Skull size of LP/ZIKV newborns is reduced.
    • Fig. S14. Summary of the results obtained with the experimental model.
    • Fig. S15. Validation of differentially expressed genes by RT-qPCR in E15 brains of Co/ZIKV and LP/ZIKV.
    • Table S1. Relationship between cases of undernutrition reported in hospitals and medical services and cases of microcephaly between 2015 and 2018.
    • Table S2. The estimated amount of daily protein (g) intake for each of the 83 interviewed mothers who have children with CZS.
    • Table S3. Relative risk of CZS in relation to protein intake.
    • Table S4. Maternal body weight (mean and SD) at the different pregnancy stages of the mouse model.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S5 (Microsoft Excel format). Differentially expressed genes between Co/ZIKV and LP/ZIKV.

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