Journal: Frontiers in Microbiology

Article Title: Deficient of glycosylation site in the envelop protein attenuated Zika virus replication in mosquito cells

doi: 10.3389/fmicb.2025.1603083

Figure Lengend Snippet: T156I mutation delays the replication of ZIKV in mosquito cells. (A,B) The wild type (WT) and T156I mutant (T156I) virus were subjected to infect mosquito C6/36 cells. The whole cell lysates were collected after 12–72 hpi to determine the expression level of E protein using western blotting assay. The GAPDH were used as the house-keeping gene for normalization. The grayscale of the bands in western blotting assay were analyzed by ImageJ software. Asterisks indicate significant differences at 24 hpi between cells infected by WT (orange column) and cells infected by T156I (green column). (C) Mosquito cells were infected by WT or T156I virus for 12 hpi and 72 hpi. E protein (green) and cell nucleic (DAPI, blue) were immuno-stained to analyze the expression of E protein. (D,E) Chloroquine and MG132 were added to the cells followed by WT or T156I infection. The whole cell lysates were collected after 24 hpi to determine the expression level of E protein using western blotting assay. The GAPDH were used for normalization. (F,G) Mosquito cells were infected by WT or T156I. The cell lysates were used to extract the total RNA and subjected to detect the mRNA level of R protein. The supernatants were used to determine the virus titer by plaque-forming assay in VeroE6 cells. (H) The morphology of plaques was scanned. All the results represent the mean value ± standard error of the mean pooled from three independent experiments with duplicated samples. Asterisks indicate significant differences between cells infected by WT (orange column) and cells infected by T156I (green column). Statistical analysis was performed with unpaired Student’s t -test, * p < 0.01, ** p < 0.005, *** p < 0.001.

Article Snippet: The membrane was blocked with 5% non-fat milk in TBST for 1 h at room temperature, followed by incubation with primary antibody against Zika virus (ZIKV) envelope protein (SinoBiological, 40543-R029) overnight at 4 °C.

Techniques: Mutagenesis, Virus, Expressing, Western Blot, Software, Infection, Staining

Journal: Frontiers in Microbiology

Article Title: Deficient of glycosylation site in the envelop protein attenuated Zika virus replication in mosquito cells

doi: 10.3389/fmicb.2025.1603083

Figure Lengend Snippet: T156I mutation potentially weaken the stability of E-dimer but not antibody receptor binding affinity (A) The molecular simulation of the T156I mutation on the structure of ZIKV E protein. The red, yellow, and blue colors represent the domain I, domain II and domain III of the E protein which mediate the formation of E-dimer. (B) The protein model was evaluated using Ramachandran plot analysis. The results indicate that 92.3% of the amino acids are located in the most favorable region, 7.1% in additional allowed regions, 0.3% in generously allowed regions, and 0.3% in disallowed regions-totaling 100%, which confirming the reliability of the E protein structural model. (C) The potential interaction patterns at the 156I residue with neighboring amino acids. Yellow stick represents the hydrogen bonds, pink stick showed the weak C–H bonds, and the gray stick represent the two sets of hydrophobic interactions with surrounding residues. (D) The HDCOK program was used to perform a global docking simulation of the binding interface and interaction mode between the 156I mutant E protein and the ZIKV neutralizing antibody EDE2. (E) Revealed that the mutant complex forms seven sets of hydrogen bonds (gray), four sets of weak C–H bonds (yellow), and two sets of hydrophobic interactions (pink). (F) The potential interactions of I156 with surrounding residues in 2D analysis.

Article Snippet: The membrane was blocked with 5% non-fat milk in TBST for 1 h at room temperature, followed by incubation with primary antibody against Zika virus (ZIKV) envelope protein (SinoBiological, 40543-R029) overnight at 4 °C.

Techniques: Mutagenesis, Binding Assay, Residue

Journal: Virology Journal

Article Title: The first trimester human placenta responds to Zika virus infection inducing an interferon (IFN) and antiviral interferon stimulated gene (ISG) response

doi: 10.1186/s12985-025-02729-3

Figure Lengend Snippet: Antibodies used in indirect Immunofluorescence detection of ZIKV and CTB antigens

Article Snippet: Rabbit anti- ZIKV Envelope Protein , Genetex, GTX133314.

Techniques: Immunofluorescence

Journal: Virology Journal

Article Title: The first trimester human placenta responds to Zika virus infection inducing an interferon (IFN) and antiviral interferon stimulated gene (ISG) response

doi: 10.1186/s12985-025-02729-3

Figure Lengend Snippet: ZIKV infects and replicates in first trimester placental explants. First trimester placental explant tissue cultures ( n = 6 patients in duplicate) were infected with ZIKV at 10 6 or 10 7 PFU/well and ( A ) ZIKV RNA genomes quantified by qRT-PCR. Data were log transformed and analysed by Ordinary Two Way ANOVA (* P < 0.05, ** P < 0.01. *** P < 0.005) or ( B ) infectious ZIKV particles in culture supernatant from infected explant tissues detected by plaque assay. Some explants that were infected at 10 6 PFU/well did not produce infectious plaques at 72hpi. The 24hpi data represents the viral inoculum, as virus was left on explants for 24 h. This data was not included in subsequent statistical analysis. Data were log transformed and analysed by Ordinary Two-Way ANOVA. (* P < 0.05, **** P < 0.0001). ( C ) Frozen sections from ZIKV infected explant tissues (96hpi) were stained for ZIKV-Envelope antigen (red), cytokeratin (green) and nuclei (DAPI). Bars represent 100 μm unless otherwise indicated. Images are representative of multiple samples analysed ( D). First trimester placental explant tissue cultures (6 patients in duplicate) were infected with DENV and at timepoints indicated DENV RNA genomes quantified by qRT-PCR including in uninfected controls. ( E ) infectious DENV particles in the culture supernatant from infected explant tissues detected by focus forming assay. All data are means± SE

Article Snippet: Rabbit anti- ZIKV Envelope Protein , Genetex, GTX133314.

Techniques: Infection, Quantitative RT-PCR, Transformation Assay, Plaque Assay, Virus, Staining, Focus Forming Assay

Journal: Virology Journal

Article Title: The first trimester human placenta responds to Zika virus infection inducing an interferon (IFN) and antiviral interferon stimulated gene (ISG) response

doi: 10.1186/s12985-025-02729-3

Figure Lengend Snippet: Zika virus infects and replicates in isolated first trimester placental trophoblasts. ( A ) Trophoblasts from first trimester placental samples were infected with ZIKV then at 24hpi and 48hpi ZIKV genome RNA was quantified using qRT-PCR, including in uninfected control cells. Data are means ± SE, n = 3 patient samples. ( B ) Infectious ZIKV in CTB culture supernatant was quantified by plaque-assay. Data are means ± SE, n = 2 patient samples in duplicate. Data were log transformed and analysed by Ordinary Two Way ANOVA (*** P < 0.005). ( C-D ) Isolated trophoblasts were fixed and stained for the CTB marker, cytokeratin-7 (red) and ZIKV-E antigen (green). ( E , F ) Detection of ZIKV genome dsRNA replication intermediates used anti-dsRNA Ab (red), cytokeratin 7 (green) and nuclei (DAPI). Bars represent 50 μm unless otherwise indicated

Article Snippet: Rabbit anti- ZIKV Envelope Protein , Genetex, GTX133314.

Techniques: Virus, Isolation, Infection, Quantitative RT-PCR, Control, Plaque Assay, Transformation Assay, Staining, Marker