Fig 1.
Astrocytes-hNS1 are a pure culture of human astrocytes.
hNS-1 cells were differentiated for 21 days. The differentiated cells were harvested and seeded in culture labware without poly-L-lysine treatment and with DMEM. Non-adherent cells (neurons and neural progenitor cells that did not differentiate) were removed by exchanging the media. Astrocytes-hNS1 cells were seeded, and cells were fixed and processed for immunofluorescence using antibodies to GFAP (astrocyte marker). Cells from different passages were used to verify that they maintained a stable karyotype using the karyotyping technique. (A) Diagram of the procedure to obtain a pure culture of astrocytes-hNS1. (B) Representative micrographs of GFAP immunofluorescence in cultured astrocytes. Scale bar 50 μm. (C) Reconstruction of the karyotype of three passages of astrocytes-hNS1.
Fig 2.
Astrocytes-hNS1 are permissible and susceptible to ZIKV infection.
(A) The astrocytes-hNS1 were seeded in 24-well plates and processed for immunofluorescence with antibodies against GFAP (green) and ZIKV envelope protein (red), and the nuclei were stained with Hoechst (blue). Scale bar 50 μm. (B) The number of cells positive for ZIKV infection was quantified at three dpi with MOI of 1 (mean percentage and standard deviation, n = 6, p = 0.031, t-test). (C) The Astrocytes were infected with ZIKV (MOI of 1), and supernatants were recovered for 5 dpi to determine viral genome production by qRT-PCR. An increase in the viral genome during the infection progress is shown (*p<0.05, **p<0.01, ***p<0.001, ANOVA with Tukey’s multiple comparisons). Each point represents the mean of 3 independent experiments with at least two duplicates, and the error bars correspond to the standard deviation. (D) Brightfield microscopy of ZIKV infected (MOI of 1) or mock-infected astrocytes-hNS1 at 5 dpi. Scale bar 100 μm. White arrows indicate cytopathic effect (E) Thin-section TEM of infected cells (MOI of 1) at 6 dpi shows the presence of viral particles inside vesicles (white arrows).
Fig 3.
ZIKV infection of astrocytes modifies the metabolic activity and the production of species reactive oxygen.
(A) Astrocytes-hNS1 were infected with ZIKV (MOI of 0.1 and 1), and at five dpi, viability was determined by indirectly measuring activity metabolism with resazurin. (B) The production of ROS was measured in mock-infected and ZIKV-infected cells at MOI of 0.1 and 1. In A and B, the data are median and interquartile range, n = 3–4, **p<0.01, ***p<0.001, Kruskal Wallis with Dunn’s post hoc. For A and B, H2O2 was used to induce cell death.
Fig 4.
Glial marker expression is altered by ZIKV infection.
The astrocytes-hNS1 culture was infected with ZIKV (MOI of 1) or mock-infected. At five dpi the cellular RNA was recovered and the expression of glial transcripts was analyzed by qRT-PCR. (A) Relative expression of GFAP in infected and uninfected cells, p = 0.002, n = 6, t-test. (B) Relative expression of EAAT1, p = 0.0006, n = 6, t-test. (C) Relative expression of GS, p = 0.023, n = 6, t-test. (D) Relative expression of EAAT2, p = 0.0006, n = 6, t-test. (E) Relative expression of CX43, p = 0.10, n = 6, U Mann Whitney test. (F) Relative expression of NMDAR, p = 0.0045, n = 6, t-test. The plotted data represent the mean and standard deviation.
Fig 5.
ZIKV infection can modify genes of the TAM family and genes involved in the lipidic metabolism.
The astrocytes-hNS1 culture was infected with either ZIKV (MOI of1) or mock-infected. At five dpi, the cellular RNA was recovered and the relative expression of the mRNAs for the receptors of the TAM family (Tyro-3, AXL, and Mertk), and APOE and PPARγ mRNAs associated with lipid metabolism were determined by qRT-PCR. (A) Tyro-3 relative expression, p = 0.025, n = 6, t-test. (B) Relative expression of AXL, p = 0.0022, n = 6, U Mann Whitney test. (C) Relative expression of Mertk, p = 0.0006, n = 6, t-test. (D) Relative expression of PPAR-γ, p = 0.24, n = 6, t-test. (E) APOE relative expression p = 0.18, n = 6, t-test. The data correspond to the mean and standard deviation.
Fig 6.
Virions generated after ZIKV infection are concentrated in vesicles.
The astrocytes-hNS1 culture was infected with ZIKV (MOI of 1) or mock-infected. At 6 dpi, the monolayers were fixed and processed for observation by transmission electron microscopy. (A) Representative micrograph of an entire astrocyte-Hns1. Cells are characterized by large multilamellar bodies (see blue arrows). (B) ZIKV-infected cells at 6dpi showed many vacuoles (see red arrows) and the multilamellar bodies were very rare in infected cellsand were small (see blue arrows). (C) ZIKV-infected cells, where virions delimited in vesicles are observed (see green arrows). (D) ZIKV-infected cells show multiple virions associated with vesicular bodies and close to the endoplasmic reticulum.
Fig 7.
ZIKV infection in astrocytes-hNS1 causes changes in the mitochondria.
The astrocyte-hNS1 culture was infected with ZIKV (MOI of 1) or mock-infected. The monolayers were fixed at 3 and 6 dpi and processed for observation by transmission electron microscopy. (A) Mock-infected cells at three dpi. (B) Mock-infected cells at six dpi. The mitochondria of the mock-infected cells show the homogeneous size and parallel cristae. (C) ZIKV-infected cells during 3 dpi showing mitochondria with concentric cristae and mitochondria with the sparse mitochondrial matrix. (D) ZIKV-infected cells 6 dpi showed decreased mitochondrial cristae and lighter electron density. (E) Quantitative analysis of the number of mitochondria per cell, the mean and standard deviation are presented, p = 0.06, ANOVA. (F) Quantitative analysis of the size of the mitochondria expressed in the mitochondrial area (μm2), the median and range are presented, p<0.0001, Kruskal Wallis test with a post hoc Dunn’s Multiple Comparison Test. In order to compare the area of the mitochondria we only analyzed organelles with the same orientation.
Fig 8.
ZIKV infection in astrocytes-hNS1 causes changes in lipid droplets (LDs).
The astrocyte-hNS1 culture was infected with ZIKV (MOI of 1) or mock-infected. At 3 and 6 dpi, the monolayers were fixed and processed for observation by transmission electron microscopy. (A) Mock-infected cells at three dpi. (B) Mock-infected cells at six dpi respectively, LDs can be observed in the cell’s cytoplasm. (C) ZIKV-infected cells (MOI of 1) at three dpi showed many LDs throughout the cytoplasm. (D) Representative micrograph of a ZIKV-infected cell at six dpi showing abnormal mitochondria and a complete absence of LDs. (E) Quantitative analysis of the number of LDs /cells, median, and range are presented, **p<0.001, ***p<0.0001, U Mann Whitney test. (F) Quantitative analysis of LDs size (area, μm2), the mean and standard deviation are presented, p = 0.10, Kruskal Wallis test, comparisons between the groups where measurements of LDs area could be determined.