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: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: (A-C) A549 cells were infected with ZIKV at an MOI of 1 for different durations (0, 12, 24, and 48 h). The cells were collected for ZIKV RNA level detected by qRT-PCR (A), PTBP1 mRNA (B), and protein (C) levels detected by qRT-PCR and Western blot, respectively. (D-F) A549 cells were infected with ZIKV at MOIs of 0, 0.25, 0.5, and 1.0 for 24 h. ZIKV RNA level was detected by qRT-PCR (A), while PTBP1 mRNA (B) and protein (C) levels were detected by qRT-PCR and Western blot, respectively. Graphs were expressed as Mean±SD, n = 3. ns, not-significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Infection, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: (A) A549 cells were infected with ZIKV at an MOI of 1 at different time points (0, 12, 24, and 48 h). Cells were collected at the corresponding time points for Western blot analysis. (B and C) A549 cells were stimulated with different doses of IOX2 (0, 20, and 40 μM) for 12 h. Cells were collected for qRT-PCR (B) and Western blot (C) analysis, respectively. (D and E) A549 cells were stimulated with IOX2 (40 μM) for 12 h, followed by infection with ZIKV (MOI = 1) for an additional 24 h. Cells were collected for qRT-PCR (D) and Western blot (E) analysis, respectively. (F and G) A549 cells were stimulated with a HIF-1α inhibitor YC-1 (20 μM) for 16 hours, followed by infection with ZIKV (MOI = 1) for another 24 h. Cells were collected for qRT-PCR (F) and Western blot (G) analysis, respectively. Graphs were expressed as Mean±SD, n = 3. ns, not-significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Infection, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: (A) LV-PTBP1 and LV-Vector control cells were subjected to Western blot analysis to detect the expression of FLAG-tagged protein. (B-D) LV-PTBP1 and LV-Vector cells were infected with ZIKV (MOI = 1) at different time points (0, 24, and 48 h). Cells were collected at the corresponding time points for qRT-PCR (B) and Western blot (C) analysis, respectively. Supernatants was subjected to TCID50, and the virus titer was calculated (D). (E) The stable expressing shPTBP1 and shNC control cells were subjected to Western blot analysis. (F-H) shPTBP1 and shNC cells were infected with ZIKV (MOI = 1) at different time points (0, 24, and 48 h). Cells were collected at the corresponding time points for qRT-PCR (F) and Western blot (G) analysis, respectively. Supernatants were subjected to TCID50, and the virus titer was calculated (H). Graphs were expressed as Mean±SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Plasmid Preparation, Control, Western Blot, Expressing, Infection, Quantitative RT-PCR, Virus

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: (A-C) shPTBP1 cells and shNC control cells were treated with different concentrations of IOX2 (0, 30, 40, 50 μM) for 12 hours and then infected with ZIKV (MOI = 1). Cells were collected 24 hours post-infection for qRT-PCR (A) and Western blot (B) analysis, respectively. Supernatants were subjected to TCID50, and the virus titer was calculated (C). Graphs were expressed as Mean±SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Control, Infection, Quantitative RT-PCR, Western Blot, Virus

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: (A) HEK293T cells were transfected with the HA-Vector (2.0, 1.75, 1.5, 1.25, and 0.75 μg) and FLAG-Vector (1.0, 0, 0, 0, 0 μg) or FLAG-NS1 (0, 0, 0, 0, 0.5 μg), or FLAG-NS5 (0, 0, 0, 0, 0.5 μg) or HA-PTBP1 (0, 0, 0.25, 0.5, 1.0) along with eGFP-C1 (0, 0.25, 0.25, 0.25, 0.25 μg) plasmids. After 30 h, the cells were collected and subjected to Western blot analysis. (B) The scheme of two main models of protein degradation: UPS (ubiquitin-proteasome system) and ALP (autophagy-lysosome pathway). (C and D) HEK293T cells (C or A549 cells (D) were transfected with 1.5 μg of each plasmid: HA-Vector, FLAG-NS1, and HA-PTBP1, along with FLAG-NS1. At 18 h post-transfection, the cells were treated with DMSO, 3-MA (1.0 mM), MG132 (2.5 μM), or NH 4 Cl (5 mM) for another 12 h. The protein levels were analyzed by Western blot. (E) A549 cells were transfected with 1.0 μg of each plasmid: HA-Vector or HA-PTBP1 along with FLAG-NS1. At 24 h post-transfection, the cells were labeled with anti-FLAG and anti-HA primary antibodies followed by corresponding fluorescent secondary antibodies. The lysosomes were stained with Lyso-Tracker, while nucleus was stained with DAPI. The images displaying NS1 (red), PTBP1 (green), Lysosome (white), and DAPI (blue) were captured under a confocal fluorescence microscope. Scale bar = 20 μm.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Ubiquitin Proteomics, Labeling, Staining, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: Upon ZIKV infection, HIF-1α signal is activated to promote PTBP1 expression. Meanwhile, ZIKV viral RNA is translated into structural and nonstructural proteins, which participate in viral replication. The HIF-1α-induced PTBP1 interacts with ZIKV NS1 and then promotes the degradation of NS1 protein in a lysosomal pathway, and finally disrupts ZIKV replication.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Infection, Expressing

Journal: Microorganisms

Article Title: Favipiravir (T-705) Protects IFNAR −/− Mice against Lethal Zika Virus Infection in a Sex-Dependent Manner

doi: 10.3390/microorganisms9061178

Figure Lengend Snippet: In vitro ribavirin and favipiravir efficacy. Vero E6 cells were infected in triplicate with four different strains of ZIKV and treated with different concentrations of favipiravir and ribavirin. Virus titration was performed to determine antiviral efficacy against ZIKV-French Polynesia, ZIKV-Paraiba, ZIKV-PRVABC59, and ZIKV-MR766 using median tissue culture infectious dose (TCID 50 ) assay. Geometric mean and standard deviation are shown.

Article Snippet: Embedded tissues were sectioned at 5 μm and dried overnight at 42 °C prior to staining with hematoxylin and eosin (H&E) or a rabbit polyclonal antibody for immunohistochemistry (IHC) against ZIKV NS2B (cat# GTX133308, Genetex, Irvine, CA, USA).

Techniques: In Vitro, Infection, Virus, Titration, Standard Deviation

Journal: Microorganisms

Article Title: Favipiravir (T-705) Protects IFNAR −/− Mice against Lethal Zika Virus Infection in a Sex-Dependent Manner

doi: 10.3390/microorganisms9061178

Figure Lengend Snippet: Ribavirin and favipiravir treatment efficacy against ZIKV-French Polynesia in IFNAR −/− mice. Female and male mice (6–9 weeks old) were infected with 1000 median lethal doses (LD 50 ) (5000 plaque-forming units (PFU)) ZIKV-French Polynesia and treated with antivirals starting 8 h post-infection and continuing treatment every 24 h until day 14. ( A ) Survival and ( B ) body weight changes for each treatment group (n = 8) and vehicle-control group (n = 8) are shown. Female and male mice were infected with 100 LD 50 (500 PFU) and treated with 300 mg/kg favipiravir starting 8 h, 24 h, or 48 h post-infection, and continued daily treatment until day 14. ( C ) Survival and ( D ) body weight changes in treated (n = 8) and vehicle-control groups (n = 8) are shown. Error bars indicate standard deviation. Statistically significant results are indicated as follows: ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Embedded tissues were sectioned at 5 μm and dried overnight at 42 °C prior to staining with hematoxylin and eosin (H&E) or a rabbit polyclonal antibody for immunohistochemistry (IHC) against ZIKV NS2B (cat# GTX133308, Genetex, Irvine, CA, USA).

Techniques: Infection, Control, Standard Deviation

Journal: Microorganisms

Article Title: Favipiravir (T-705) Protects IFNAR −/− Mice against Lethal Zika Virus Infection in a Sex-Dependent Manner

doi: 10.3390/microorganisms9061178

Figure Lengend Snippet: Sex differences in favipiravir-treated and ZIKV-infected IFNAR −/− mice. Female and male mice (6–9 weeks old) were infected with 100 LD 50 (500 PFU) ZIKV-French Polynesia and treated with 300 mg/kg favipiravir starting 8 h post-infection and continuing treatment every 24 h until day 14. ( A ) Survival and ( B ) body weight changes for each treatment group (n = 8) and vehicle-control group (n = 8) are shown. Error bars indicate standard deviation. On days 3 and 7 after infection, blood and tissue samples were taken to determine ( C ) ZIKV titer (n = 4) by using median tissue culture infectious dose (TCID 50 ) assay and ( D ) ZIKV load (n = 4). Geometric mean is depicted. Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01.

Article Snippet: Embedded tissues were sectioned at 5 μm and dried overnight at 42 °C prior to staining with hematoxylin and eosin (H&E) or a rabbit polyclonal antibody for immunohistochemistry (IHC) against ZIKV NS2B (cat# GTX133308, Genetex, Irvine, CA, USA).

Techniques: Infection, Control, Standard Deviation

Journal: Microorganisms

Article Title: Favipiravir (T-705) Protects IFNAR −/− Mice against Lethal Zika Virus Infection in a Sex-Dependent Manner

doi: 10.3390/microorganisms9061178

Figure Lengend Snippet: Pathological analysis of tissue samples from favipiravir-treated, ZIKV-infected IFNAR −/− mice. Female and male mice (6–9 weeks old) were infected with 100 LD 50 (500 PFU) ZIKV-French Polynesia and treated with 300 mg/kg favipiravir starting 8 h post-infection and continuing treatment every 24 h until day 14. Tissue samples were collected from four mice per group on day 7 post-infection. ( A ) Tissues were scored for pathology using the following scoring system: 0 = no lesions; 1 = small number of necrotic cells; 2 = moderate necrosis; 3 = significant necrosis; 4 = coalescing necrosis; 5 = diffuse necrosis. Error bars indicate standard deviation. ( B ) Brain tissue fixed with formalin was stained with hematoxylin and eosin (H&E) and imaged at 100× or stained for ZIKV NS2B antigen using immunohistochemistry (IHC) and imaged at 400× magnification. Arrows indicate meningoencephalitis (perivascular cuffing); arrowheads indicate gliosis.

Article Snippet: Embedded tissues were sectioned at 5 μm and dried overnight at 42 °C prior to staining with hematoxylin and eosin (H&E) or a rabbit polyclonal antibody for immunohistochemistry (IHC) against ZIKV NS2B (cat# GTX133308, Genetex, Irvine, CA, USA).

Techniques: Infection, Standard Deviation, Staining, Immunohistochemistry